Silencing of HNF4A-P2 Isoforms with siRNA to Improve Hepatocyte Function in Liver Failure

ABSTRACT

Provided herein are antisense agents for knocking down, inhibiting, or silencing expression of an HNF4α-P2 isoform mRNA in a cell or a patient. Also provided are methods of knocking down, inhibiting, or silencing expression of an HNF4α-P2 isoform mRNA in a cell or a patient and methods of treating a patient with liver disease, liver damage, liver inflammation, or liver failure, such as acute liver failure, ALD, AH, or ACLF.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of United States Provisional PatentApplication No. 62/753,299, filed Oct. 31, 2018 and U.S. ProvisionalPatent Application No. 62/757,944, filed Nov. 9, 2018, each of which isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under Grant Nos.AA021908 and AA026972 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 1907658_ST25.txt. The size of thetext file is 60,883 bytes, and the text file was created on Oct. 28,2019.

Liver-related mortality has increased in the last decade, partially dueto the higher incidence of addictions in the form of alcohol-relatedcirrhosis. The prognosis of alcohol-related liver disease (ALD) dependson the development of liver failure, mainly in the form of alcoholichepatitis (AH). The burden of AH has increased in many countries andrepresents an important public health problem. The genetic andepigenetic factors involved in the development of AH in heavy drinkersare not well known. Genome-wide association studies (GWAS) have shownthat variations in PNPLA3 (patatin like phospholipase domain containing3), MBOAT7 (membrane-bound O-acetyltransferase domain-containing protein7), and TM6SF2 (transmembrane 6 superfamily member 2) loci confer riskfor alcohol-related cirrhosis, but the association of specific loci withAH is unknown. Because alcohol abuse has been associated with DNAmethylation changes in humans and epigenetic dysregulation inexperimental liver injury, it is conceivable that epigenetic factorsplay a role in AH. Liver failure in the setting of AH was traditionallyconsidered to be secondary to a flare in intrahepatic inflammation.Consequently, therapies have been directed towards decreasinginflammatory mediators (e.g., prednisolone), with limited efficacy.Bilirubinostasis, inefficient regeneration of hepatocytes and acompensatory ductular reaction may play a pathogenic role in AH.However, the mechanisms of liver failure in the setting of AH remainobscure.

Liver failure is a common cause of morbidity and mortality. There are notargeted therapies to improve liver function. Alcoholic hepatitis (AH),a common cause of liver failure, is a life-threatening conditioncharacterized by profound hepatocellular dysfunction for which targetedtreatments are urgently needed.

SUMMARY

Provided herein is a method of treating a patient having liver damage orliver failure. The method comprises knocking down or inhibitingexpression of a hepatocyte nuclear factor 4 alpha mRNA transcribed fromits P2 promoter (HNF4α-P2 isoform mRNA) in the patient, or reducingactivity of the protein encoded by the HNF4α-P2 isoform mRNA, therebytreating the liver damage or liver failure in the patient.

Also provided herein is a method of knocking down expression of anHNF4α-P2 isoform mRNA in a cell. The method comprises contacting thecell with an RNAi agent for selectively knocking down expression of anHNF4α-P2 isoform mRNA, in an amount effective to reduce production ofthe protein product of the HNF4α-P2 isoform mRNA in a cell.

Also provided herein is an RNAi agent targeting exon 1D of HFN4α.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A and 1B. Liver transcriptome analysis in patients with ALD showdeficient predicted activity of LETFs in AH. (FIG. 1A) Transcriptionfactor footprint analysis between early ASH and AH patient livers. Mostsignificant differentially expressed (DE) genes in the comparisonbetween early ASH and AH patients. Predicted activation by IPA expressedas ZS (left) and motif enrichment by Opossum as ZS and FS (right). Left,TF binding motif enrichment by Opossum analysis is expressed as ZS andFisher test Score (FS). Right, predicted activation by IPA analysisexpressed as ZS. (FIG. 1B) Selected target genes of HNF4a identified byIPA analysis. Fold Change (FC) in Normal vs Early ASH and between EarlyASH and AH are presented. All genes in (b) had a FDR <10⁻⁶ in DEanalysis.

FIGS. 2A-2S. Fetal HNF4a-P2 isoform is increased in patients withAH-related liver failure and its inhibition increases HNF4a-P1expression and function. (FIG. 2A) Combined graph showing levels ofbilirubin, INR, and albumin levels in serum along ALD progression(values expressed as Mean+/−SEM) and HNF4a footprint for each comparisonagainst control group (values expressed as ZScore from Ingenuity PathwayAnalysis). (FIG. 2B) Scheme of HNF4A gene fetal and adult isoformsstructure and protein variants. During embryonic development, P2promoter is used and alternative splicing of the first exon is produced,originating the fetal isoforms a7-12, who lack the AF-1 domain in theN-terminal of the protein. Alternative splicing of the 10th exonoriginates C-terminal variants (not presented here). (FIG. 2C) Real-TimePCR of HNF4A-P1 and P2 dependent isoforms, and of the lncRNAN HNF4A-AS1,who shares promoter with HNF4A-P1 of the same patients in FIGS. 1A and1B. (FIG. 2D) Immunohistochemical detection of adult and fetal HNF4Aprotein variants (hereinafter P1 and P2) in patients with AH (n=9), andcontrols (n=9), using specific antibodies against the N-terminalresidues. (FIG. 2E) Semi-quantitative assessment of IHC signal for eachantibody for nuclear staining. (FIGS. 2F-2I) Primary human hepatocyteswere cultured in hepatocyte growth media and collected at baseline andat 24 and 48 h (n=3 for each time point). Quantitative RT-PCR of (FIG.2F) HNF4a-P1 and P2 isoforms, (FIG. 2G) phosphoenol-pyruvatecarboxykinase (PCK1) and (FIG. 2H) albumin (ALB). (FIG. 2I) Pearsoncorrelation index among mRNA levels of HNF4A-P1 dependent genes (PCK1,CYP2E1, ALB) and of non-hepatocyte genes related to ductular metaplasia(EPCAM, KRT7), epithelial-to-mesenchymal transition (VIM) and hepatocytepro-inflammatory genes (IL8). (FIGS. 2J and 2K) HepG2 cells weretransfected with plasmids encoding P1 (HNF4a2) and P2 (HNF4a8) variants.P1 was maintained at the same dose while P2 was increased as indicated.RNA was extracted 12 h and 24 h after transfection (n=3 for each group).qPCR of (FIG. 2J) HNF4a-P2 isoform and (FIG. 2K) PCK1. (FIGS. 2L-2Q)HepG2 cells were transfected with siRNA complementary to the first exon(1E) of HNF4a-P2 isoforms (n=3 for each group), and RNA and protein wasextracted at 48 h after transfection. (1-m) levels of HNF4a-P2 (FIG. 2L)mRNA and (FIG. 2M) protein. (FIGS. 2N-2Q) mRNA levels of (FIG. 2N)HNF4a-P1, (FIG. 2O) HNF4A-AS1 lncRNA and of HNF4a-P1 targets (FIG. 2P)biosynthetic-related genes PCK1, ALB, F7 and (FIG. 2Q) bile acidsynthesis and transport genes CYP7A1, BSEP, and CYP27A1. (FIGS. 2R and2S) Primary hepatocytes were silenced with siRNA-HNF4A-P2 andsupernatant was collected 48 h after transfection (n=3 for each group).Total bile acids (FIG. 2R) and glucose production (FIG. 2S) werequantified. Significance was determined by unpaired, two-tailedStudent's test in FIGS. 2A and 2C, by Fisher exact probability test inFIG. 2D and by two-tailed Mann-Whitney U test in FIGS. 2F-2H, 2J-2K, 2L,and 2N-2S: *P<0.05, **P<0.01, ***P<0.001. Pearson Correlation test wasapplied in FIG. 2I, values inside the box indicate R coefficient foreach correlation. For box-and-whisker plots: perimeters, 25th-75thpercentile; midline, median; whiskers, minimum to maximum values;individual data points are represented. Gene expression are presented asrelative values normalized to the mean of the control. Western blot in(FIG. 2M) was performed in different experiments in HepG2 (n=3) and inHep3B (n=3) cells, representative blot is presented here.

FIGS. 3A-3F. TGFb1 is a major transcriptome regulator in AH,downregulates HNF4a partially by inducing HNF4a-P2 and is modulated byPPARg agonists and corticosteroids. (FIGS. 3A-3F) HepG2 cells weretransfected with siRNA complementary to the first exon (1E) of HNF4a-P2isoforms, and RNA and nuclear proteins were extracted at 48 h aftertransfection and 8 h (RNA) or 24 h (Nuclei) after TGFb1 treatment (5ng/ml) (n=4 for each condition). (FIGS. 3A and 3B) levels of HNF4a-P2(FIG. 3A) mRNA and (FIG. 3B) protein. (FIGS. 3C and 3D) mRNA levels ofHNF4a-P1 targets (FIG. 3C) biosynthetic-related genes PCK1, ALB, F7 and(FIG. 3D) bile acid synthesis and transport genes CYP7A1, BSEP. (FIGS.3E and 3F) Primary human hepatocytes were silenced with siRNA-HNF4a-P2and supernatant was collected 48 h after transfection and 8 h afterTGFb1 treatment (5 ng/ml) (n=3 for each condition). Total bile acids(FIG. 3E) and Glucose production (FIG. 3F) were quantified. Significancewas determined by two-tailed Mann-Whitney U test in a, c, d, and e:*P<0.05, **P<0.01. For box-and-whisker plots: perimeters, 25th-75thpercentile; midline, median; whiskers, minimum to maximum values;individual data points are represented. Gene expression is presented asrelative values normalized to the mean of the control.

FIGS. 4A and 4B (continuous) provide an exemplary nucleotide sequencefor an HNF4A transcript variant (NM_175914.4 Homo sapiens hepatocytenuclear factor 4 alpha (HNF4A), transcript variant 5, mRNA (SEQ ID NO:1)).

FIGS. 5A, 5B, and 6 provide non-limiting additional examples of HNFAisoforms initiating from the P2 promoter, including HNF4alpha7(NM_001030003.2, FIGS. 5A and 5B, continuous, SEQ ID NO: 2) andHNF4alpha9 (NM_001030004.2, FIG. 6, SEQ ID NO: 3).

FIG. 7A provide exemplary additional oligomers useful as RNAi agents forinhibition of HNF4α-P2 isoforms based on GenBank accession No.NM_175914.4. FIG. 7B provides a legend for nucleotide modificationsshown in FIG. 7A (SEQ ID NOS: 4-37).

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values. For definitions provided herein, those definitionsrefer to word forms, cognates and grammatical variants of those words orphrases. As used herein “a” and “an” refer to one or more.

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, are open ended and do not exclude the presence ofother elements not identified. In contrast, the term “consisting of” andvariations thereof is intended to be closed, and excludes additionalelements in anything but trace amounts.

As used herein, the term “patient” or “subject” refers to members of theanimal kingdom including but not limited to human beings and “mammal”refers to all mammals, including, but not limited to human beings.

As used herein, the “treatment” or “treating” of liver disease, liverdamage, liver inflammation, or liver failure, such as acute liverfailure, ALD, AH, or Acute-on-Chronic Liver Failure (ACLF), meansadministration to a patient by any suitable dosage regimen, procedureand/or administration route of a composition, device, or structure withthe object of achieving a beneficial or desirable clinical/medicalend-point, including but not limited to, preventing, reducing, and/oreliminating any symptom of liver disease, liver damage, liver cirrhosis,liver inflammation, or liver failure, such as acute liver failure, ALD,AH, or ACLF, such as liver damage or liver inflammation. An amount ofany agent, administered by any suitable route, effective to treat apatient is an amount capable of preventing, reducing, and/or eliminatingany symptom of liver disease, liver damage, liver inflammation, or liverfailure, such as acute liver failure, ALD, AH, or ACLF, such as liverdamage or liver failure in a patient. Liver damage and Liver failure, aswell as, ALD, AH, or ACLF, are accompanied by, and can be identified byincreased blood bilirubin levels, increased prothrombin time, and/orreduced serum albumin levels, which can be tested by well-known clinicalassays. As such, “treatment” of liver injury, liver damage, liverinflammation, liver failure, cirrhosis, ALD, AH, or ACLF, may result indecreased blood bilirubin levels, decreased prothrombin time, orincreased serum albumin. Clinical assay results, such as blood bilirubinlevels, decreased prothrombin time, or increased serum albumin can besaid to “normalize” when such levels approach or enter a normal orhealthy range for a patient.

Provided herein is a method of treating liver disease, liver damage, orliver failure, such as acute liver failure, ALD, AH, or ACLF in apatient that comprises selectively decreasing expression of HNF4A-P2(fetal) isoform, preferably without significantly decreasing expressionof the HNF4A-P1 (mature) isoform in a patient, e.g., in a patient'sliver. To selectively decrease expression of the HNF4A-P2 isoform,expression of that isoform may be knocked down or knocked out in somemanner. There are a number of ways to decrease expression or activity ofa gene in a patient, including, for example, and without limitation: RNAinterference, antisense technology, and inhibition of thetranscriptional activation activity of HNF4A-P2 isoform through use of,e.g., small molecules or agents that interfere with activity of theHNF4A-P2 isoform, such as decoys, binding reagents, antagonists, etc.However, a goal of the method provided herein is to reduce one specificisoform, and, as such, only that isoform may be targeted. As shownherein, RNA interference (RNAi) is one method by which expression of theHNF4A-P2 isoform can be specifically knocked down without significantlyknocking down expression of the HNF4A-P1 isoform. Treatment of a patientresults in a decrease in one or more symptoms of liver disease, liverdamage, liver failure, such as acute liver failure, ALD, AH, or ACLF ina patient, such as liver damage and liver inflammation. Suitable markersfor successful treatment include, without limitation, decreased ornormalized blood bilirubin levels, decreased or normalized prothrombintime, or increased or normalized serum albumin in a patient with liverdamage, liver failure, liver inflammation, liver disease, cirrhosis,ALD, AH, or ACLF.

The compositions described herein can be administered by any effectiveroute, such as parenteral, e.g., intravenous, intramuscular,subcutaneous, intradermal, etc., formulations of which are describedbelow and in the below-referenced publications, as well as arebroadly-known to those of ordinary skill in the art.

Suitable dosage forms may include single-dose, or multiple-dose vials orother containers, such as medical syringes, containing a compositioncomprising an active ingredient useful for treatment of liver disease,liver damage, liver failure, such as acute liver failure, ALD, AH, orACLF, as described herein.

Drug products, or pharmaceutical compositions comprising an active agent(e.g., drug), for example, an active agent that decreases HNF4A-P2isoform activity, may be prepared by any method known in the art ofpharmacy, for example, by bringing into association the activeingredient with the carrier(s) or excipient(s). As used herein, a“pharmaceutically acceptable excipient”, “carrier” or “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable excipients include one or moreof water, saline, phosphate buffered saline, dextrose, glycerol,ethanol, and the like, as well as combinations thereof. In many cases,it may be preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Pharmaceutically acceptable carriers may further compriseminor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the active agent. The active agent may be prepared witha carrier that will protect the compound against rapid release, such asa controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used in delivery systems, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for the preparation ofsuch formulations are broadly-known to those skilled in the art.

Additionally, active agent-containing compositions may be in a varietyof forms. The preferred form depends on the intended mode ofadministration and therapeutic application, which will in turn dictatethe types of carriers/excipients. Suitable forms include, but are notlimited to, liquid, semi-solid and solid dosage forms.

Pharmaceutical formulations adapted for oral administration may bepresented, for example and without limitation, as discrete units such ascapsules or tablets; powders or granules; solutions or suspensions inaqueous or non-aqueous liquids; edible foams or whips; or oil-in-waterliquid emulsions or water-in-oil liquid emulsions. In certainembodiments, the active agent may be contained in a formulation suchthat it is suitable for oral administration, for example, by combiningthe active agent with an inert diluent or an assimilable edible carrier.The active agent (and other ingredients, if desired) may also beenclosed in a hard or soft shell gelatin capsule, compressed intotablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Pharmaceutical formulations adapted for transdermal administration maybe presented, for example and without limitation, as discrete patchesintended to remain in intimate contact with the epidermis of therecipient for a prolonged period of time or electrodes for iontophoreticdelivery.

Pharmaceutical formulations adapted for topical administration may beformulated, for example and without limitation, as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays,aerosols, or oils. Formulations for topical administration of nucleicacids can include sterile and non-sterile aqueous solutions, non-aqueoussolutions in common solvents such as alcohols, or solutions of thenucleic acids in liquid or solid oil bases. The solutions can alsocontain buffers, diluents, and other suitable additives.Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can be used.

Pharmaceutical formulations adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size, forexample, in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e., by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable formulations wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalationinclude, without limitation, fine particle dusts or mists which may begenerated by means of various types of metered dose pressurizedaerosols, nebulizers, or insufflators. In the context of delivery of theactive agents described herein by inhalation, inhalation drug products,such as metered-dose inhalers, as are broadly-known in thepharmaceutical arts, are used. Metered dose inhalers are configured todeliver a single dose of an active agent per actuation, though multipleactuations may be needed to effectively treat a given patient.

Pharmaceutical formulations adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain, for example and without limitation, anti-oxidants, buffers,bacteriostats, lipids, liposomes, emulsifiers, also suspending agentsand rheology modifiers. The formulations may be presented in unit-doseor multi-dose containers, for example, sealed ampoules and vials, andmay be stored in a freeze-dried (lyophilized) condition requiring onlythe addition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules, andtablets.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. For example, sterile injectablesolutions may be prepared by incorporating the active agent in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, typical methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

A “therapeutically effective amount” refers to an amount of a drugproduct or active agent effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic result. An “amounteffective” for treatment of a condition is an amount of an active agentor dosage form, such as a single or multiple metered doses from ametered-dose inhaler, effective to achieve a determinable end-point. The“amount effective” is preferably safe—at least to the extent thebenefits of treatment outweighs the detriments and/or the detriments areacceptable to one of ordinary skill and/or to an appropriate regulatoryagency, such as the U.S. Food and Drug Administration. A therapeuticallyeffective amount of an active agent may vary according to factors suchas the disease state, age, sex, and weight of the individual, and theability of the active agent to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the active agent are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount may be less thanthe therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time, or the composition may be administered continuously or in apulsed fashion with doses or partial doses being administered at regularintervals, for example, every 10, 15, 20, 30, 45, 60, 90, or 120minutes, every 2 through 12 hours daily, or every other day, etc. Thedosage may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. In some instances, it may beespecially advantageous to formulate parenteral or inhaled compositionsin dosage unit form for ease of administration and uniformity of dosage.The specification for the dosage unit forms of the invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic or prophylacticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

By “target-specific” or reference to the ability of one compound to bindanother target compound specifically, it is meant that the compoundbinds to the target compound to the exclusion of others in a givenreaction system, e.g., in vitro, or in vivo, to acceptable tolerances,permitting a sufficiently specific diagnostic or therapeutic effectaccording to the standards of a person of skill in the art, a medicalcommunity, and/or a regulatory authority, such as the U.S. Food and DrugAgency (FDA). The active agent described herein may be target specificin the context of targeting HNF4A-P2 isoform, down-regulating HNF4A-P2isoform activity, and effectively treating liver disease, liver damage,or liver failure, such as acute liver failure, ALD, AH, or ACLF, asdescribed herein.

A “gene” is a sequence of DNA or RNA which codes for a molecule, such asa protein or a functional RNA that has a function. Nucleic acids arebiopolymers, or small biomolecules, essential to all known forms oflife. They are composed of nucleotides, which are monomers made of threecomponents: a 5-carbon sugar, a phosphate group and a nitrogenous base.If the sugar is a simple ribose, the polymer is RNA; if the sugar isderived from ribose as deoxyribose, the polymer is DNA. DNA uses thenitrogenous bases guanine, thymine, adenine, and cytosine. RNA uses thenitrogenous bases guanine, uracil, adenine, and cytosine.

Complementary refers to the ability of polynucleotides (nucleic acids)to hybridize to one another, forming inter-strand base pairs. Base pairsare formed by hydrogen bonding between nucleotide units in antiparallelpolynucleotide strands. Complementary polynucleotide strands can basepair (hybridize) in the Watson-Crick manner (e.g., A to T, A to U, C toG), or in any other manner that allows for the formation of duplexes.When using RNA as opposed to DNA, uracil rather than thymine is the basethat is considered to be complementary to adenosine. Two sequencescomprising complementary sequences can hybridize if they form duplexesunder specified conditions, such as in water, saline (e.g., normalsaline, or 0.9% w/v saline) or phosphate-buffered saline), or underother stringency conditions, such as, for example and withoutlimitation, 0.1×SSC (saline sodium citrate) to 10×SSC, where 1×SSC is0.15M NaCl and 0.015M sodium citrate in water. Hybridization ofcomplementary sequences is dictated, e.g., by salt concentration andtemperature, with the melting temperature (Tm) lowering with increasedmismatches and increased stringency. Perfectly matched sequences aresaid to be fully complementary, or have 100% sequence identity (gaps arenot counted and the measurement is in relation to the shorter of the twosequences). In one example, a sequence that “specifically hybridizes” toanother sequence, does so in a hybridization solution containing 0.5Msodium phosphate buffer, pH 7.2, containing 7% SDS, 1 mM EDTA, and 100mg/ml of salmon sperm DNA at 65° C. for 16 hours and washing twice at65° C. for twenty minutes in a washing solution containing 0.5×SSC and0.1% SDS, or does so under conditions more stringent than 2×SSC at 65°C., for example, in 0.2×SSC at 55° C. A sequence that specificallyhybridizes to another typically has at least 80%, 85%, 90%, 95%, OR 99%sequence identity with the other sequence.

“Expression” of a gene refers to the conversion of a DNA sequence of agene, e.g., the HNF4A gene, to an active, mature gene product such as apolypeptide/protein, or a functional nucleic acid, and includes, forexample, transcription, post-transcriptional modification (e.g.,splicing) translation, and post-translational processing and/ormodification of a protein. Expression of a gene can be reduced by anyeffective mechanism at any stage of the gene expression process, such asby affecting transcriptional activation, transcription,post-transcriptional RNA processing, translation, and post-translationalprocessing or modification. Expression of an mRNA, such as the HNF4α-P2isoform mRNA, described herein refers to, without limitation, any aspectof transcription of, splicing of, translation of, and post-translationalprocessing, stability, and activity of the protein product of the mRNA,e.g., the protein product of the HNF4α-P2 isoform mRNA of the HFN4Agene. Activity of a gene product may be decreased not only by decreasingexpression of the active protein product, but by affecting the matureprotein product, such as by blocking, decoying, or otherwise interferingwith the binding of the active product, or a complex containing theactive product, to prevent its activity.

Transcription is the process by which the DNA gene sequence istranscribed into pre-mRNA (messenger RNA). The steps include: RNApolymerase, together with one or more general transcription factors,binds to promoter DNA. Transcription factors (TFs) are proteins thatcontrol the rate of transcription of genetic information from DNA tomessenger RNA, by binding to a specific DNA sequence (i.e., the promoterregion). The function of TFs is to regulate genes in order to make surethat they are expressed in the right cell at the right time and in theright amount throughout the life of the cell and the organism. Thepromoter region of a gene is a region of DNA that initiatestranscription of that particular gene. Promoters are located near thetranscription start sites of genes, on the same strand, and often, butnot exclusively, are upstream (towards the 5′ region of the sensestrand) on the DNA. Promoters can be about 100-1000 base pairs long.Additional sequences and non-coding elements can affect transcriptionrates. If the cell has a nucleus (eukaryotes), the RNA is furtherprocessed. This includes polyadenylation, capping, and splicing.Polyadenylation is the addition of a poly(A) tail to a messenger RNA.Capping refers to the process wherein the 5′ end of the pre-mRNA has aspecially altered nucleotide. During RNA splicing, pre-mRNA is edited.Specifically, during this process introns are removed and exons arejoined together. The resultant product is known as mature mRNA. The RNAmay remain in the nucleus or exit to the cytoplasm through the nuclearpore complex.

RNA levels in a cell, e.g., mRNA levels, can be controlledpost-transcriptionally. Native mechanisms, including: endogenous genesilencing mechanisms, interference with translational mechanisms,interference with RNA splicing mechanisms, and destruction of duplexedRNA by RNAse H, or RNAse H-like, activity. As is broadly-recognized bythose of ordinary skill in the art, these endogenous mechanisms can beexploited to decrease or silence mRNA activity in a cell or organism ina sequence-specific, targeted manner. Antisense technology typicallyinvolves administration of a single-stranded antisense oligonucleotide(ASO) that is chemically-modified, e.g., as described herein, forstability, and is administered in sufficient amounts to effectivelypenetrate the cell and bind in sufficient quantities to target mRNAs incells. RNA interference (RNAi) harnesses an endogenous and catalyticgene silencing mechanism, which means that once, e.g., a microRNA, ordouble-stranded siRNA has been delivered, they are efficientlyrecognized and stably incorporated into the RNA-induced silencingcomplex (RiSC) to achieve prolonged gene silencing. Both antisensetechnologies and RNAi have their strengths and weaknesses, either may beused effectively to decrease or silence expression of a gene or geneproduct, such as HNF4A-P2 isoform (see, e.g., Watts, J. K., et al. Genesilencing by siRNAs and antisense oligonucleotides in the laboratory andthe clinic (2012) 226(2):365-379).

As used herein, “agent” or “RNAi agent”, when used in the context of anantisense, RNAi, or ribozyme, or other single-stranded ordouble-stranded RNA interfering nucleic acids, refers not only to RNAstructures, but effective nucleic acid analog structures. In antisenseand RNAi technologies, use of RNA poses significant delivery issues dueto the lability of RNA molecules. As such, RNA is commonlychemically-modified to produce nucleic acid analogs, not only to enhancestability of the nucleic acid molecules, but often resulting inincreased binding affinity, and with reduced toxicity. Suchmodifications are broadly-known to those of ordinary skill in the art,and are available commercially (see, e.g., Corey, D. R., Chemicalmodification: the key to clinical application of RNA interference?(2007) J Clin Invest. 117(12):3615-3622, also describing RNAi, andUnited States Patent Application Publication No. 2017/0081667).Non-limiting examples of modifications to the nucleic acid structure innucleic acid analogs include: modifications to the phosphate linkage,such as phosphoramidates or phosphorothioates; sugar modification, suchas 2′-O, 4′-C methylene bridged, locked nucleic acid (LNA), 2′-methoxy,2′-O-methoxyethyl (MOE), 2′-fluoro, S-constrained-ethyl (cEt), andtricyclo-DNA (tc-DNA); and non-ribose structures, such asphosphorodiamidate morpholino (PMO) and peptide-nucleic acids (PNA).

In addition to those HNF4A-P2 isoform-active RNAi agents describedherein, antisense reagents (ASOs), other RNAi agents, ribozyme reagents,and other nucleic acid-based methods of reducing gene expression, may bedesigned and tested, based on known sequences of HNF4A P2 and P1 isoformRNAs and gene structure (exemplary sequences are provided herein, andthe HNF4A gene is well-studied). Based on the present disclosure, one ofordinary skill can design, and/or produce an active agent capable ofknocking down HNF4A-P2 isoform expression. Of note, a number ofpublications describe algorithms for generating candidate iRNAsequences, and publically-available software can be used to implementthose algorithms. As such, typically, one only needs to enter a mRNAsequence into a calculator to produce candidate iRNAs. In the presentcase, because the HNF4A-P2 isoform target shares significant sequenceidentity with the HNF4A-P1 isoform, those methods and software platformscould not be used to generate candidate sequences. As described herein,HNF4A is transcribed from two different promotors, one expressed in thefetus, and one in adults. As such, the HNF4A P1 and P2 transcriptscomprise different 5′ ends, with different 5′ UTRs (untranslatedregions) and different 5′ sequences in their ORF (open-reading frame)upstream from the first splice site. The full sequence of the HNF4A-P2isoform could not be used to generate an iRNA candidate within the 5′sequences unique to the P2 isoform. Also, the sequences specific to theP2 isoform could not be used by themselves to generate an effective iRNAsequence. Only when the sequences specific to the P2 isoform werecombined with partial sequences of the sequences common to both the P1and P2 isoform, could a candidate iRNA sequence bedetermined—hybridizing to the unique P2 sequence. Exemplary sequences ofiRNA specific to the HNF4A-P2 isoform are provided below.

By “reducing activity” of a gene or gene product, e.g., an HNF4α-P2isoform mRNA, it is meant, by any method of specifically decreasing,suppressing, or silencing expression of the gene, decreasing activity ofthe gene product, and/or reducing available levels of the gene productin the patient. Activity of an HNF4α-P2 isoform mRNA can be reduced,e.g., by use of antisense nucleic acids, or by use of RNAi agents.Activity of an HNF4α-P2 isoform mRNA also can be reduced by antagonism,or otherwise blocking or interfering with the activity of an HNF4α-P2isoform mRNA, or by mutation. Available levels of the gene product maybe reduced in a patient, for example, either systemically, or locally inthe liver, by binding of the an HNF4α-P2 isoform mRNA with an HNF4α-P2isoform mRNA-binding reagent, such as an antibody, and antibodyfragment, or an anti-HNF4α-P2 isoform mRNA paratope-containingpolypeptide compositions, or a nucleic acid decoy comprising an HNF4α-P2isoform mRNA nucleotide binding sequence motif acting to competitivelybind an HNF4α-P2 isoform mRNA.

By decreasing, down-regulating, or knocking down an HNF4α-P2 isoformmRNA expression or activity, it is meant any action that results inlower activity of an HNF4α-P2 isoform mRNA in a cell orpatient—typically by use of a therapeutic agent. Useful therapeuticagents include, without limitation, antisense or RNAi agents; bindingreagents, such as antibodies (including antibody fragments orantibody-based polypeptide ligands), and aptamers; antagonists; decoys;and peptide-based therapies.

The terms “HNF4α” and “hepatocyte nuclear factor 4 alpha” refer to anuclear transcription factor which binds DNA as a homodimer. The encodedprotein controls the expression of several genes, including hepatocytenuclear factor 1 alpha, a transcription factor which regulates theexpression of several hepatic genes. This gene may play a role indevelopment of the liver, kidney, and intestines. Mutations in this genehave been associated with monogenic autosomal dominantnon-insulin-dependent diabetes mellitus type I. Alternative splicing ofthis gene results in multiple transcript variants encoding severaldifferent isoforms, including the P2 isoforms, and having an amino acidsequence from any vertebrate or mammalian source, including, but notlimited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine,primate, monkey, and guinea pig, unless specified otherwise. The termalso refers to fragments and variants of native HNF4α that maintain atleast one in vivo or in vitro activity of a native HNF4α. The termencompasses full-length unprocessed precursor forms of HNF4α as well asmature forms resulting from, e.g., post-translational processing.

The sequence of an exemplary human HNF4α mRNA transcript correspondingto an HNF4α-P2 isoform is provided herein as SEQ ID NO: 1. The “targetsequence” within that HNF4α-P2 isoform mRNA sequence refers to acontiguous portion of the nucleotide sequence of human HNF4α mRNAmolecule formed during the transcription of an HNF4α gene that ispresent in HNF4α-P2 isoform mRNA, but not in HNF4α-P1 isoform mRNA, andtherefore includes at least a portion of the sequence of exon 1D,including the 5′ UTR and 5′ portion of the ORF, for example and withoutlimitation, extending from, or including contiguous sequences of fromabout 15-30 bases of bases 1-53 of SEQ ID NO: 1 (underlined in FIG. 4)and having the sequence: 5′-GGCCATGGTC AGCGTGAACG CGCCCCTCGG GGCTCCAGTGGAGAGTTCTT ACG-3′ (SEQ ID NO: 1, bases 1-58). This sequence is anexemplary human sequence, and is taken from Genbank Accession No.NM_175914.4 (Homo sapiens hepatocyte nuclear factor 4 alpha (HNF4A),transcript variant 5, mRNA, FIG. 4). Additional exemplary HNF4α-P2isoforms initiating from the P2 promoter include HNF4alpha7(NM_001030003.2, FIG. 5) and HNF4alpha9 (NM_001030004.2, FIG. 6). Thetarget portion of the sequence may be at least long enough to serve as asubstrate for iRNA-directed cleavage at or near that portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an HNF4α gene, e.g., from the HNF4α-P2 isoform promoter. The targetsequence may extend up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases 3′ toexon 1D, e.g., is a contiguous sequence of at least about 15 baseswithin GGCCATGGTCAGCGTGAACGCGCCCCTCGGGGCTCCAGTGGAGAGTTCTTAC GACACGTCCCC(SEQ ID NO: 1, bases 1-63). The target sequence may be 15 or morecontiguous bases of GCTCCAGTGGAGAGTTCTTACGACACG (SEQ ID NO: 1, bases32-58), such as GCTCCAGTGGAGAGTTCTT (SEQ ID NO: 1, bases 32-50),CTCCAGTGGAGAGTTCTTA (SEQ ID NO: 1, bases 33-51), TCCAGTGGAGAGTTCTTAC(SEQ ID NO: 1, bases 34-52), or GGAGAGTTCTTACGACAC (SEQ ID NO: 1, bases40-57). All nucleotide sequences are shown in a 5′ to 3′ directionunless indicated to the contrary. FIGS. 7A and 7B provide exemplaryadditional oligomers useful as RNAi agents for inhibition of HNF4α-P2isoforms based on GenBank accession No. NM_175914.4. The target sequencemay be present in HNF4α-P2 isoforms and not in HNF4α-P1 isoforms, andthus can selectively decrease or knock down HNF4α-P2 isoform mRNAs andnot HNF4α-P1 isoform mRNAs, at least not to any significant extent inthe context of the methods described herein.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine, and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacementmoiety. The skilled person is well aware that guanine, cytosine,adenine, and uracil can be replaced by other moieties withoutsubstantially altering, or improving in the context of RNA interference,the base pairing properties of an oligonucleotide comprising anucleotide bearing such replacement moiety. For example, withoutlimitation, a nucleotide comprising inosine as its base can base pairwith nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA by a nucleotide containing, forexample, inosine. In another example, adenine and cytosine anywhere inthe oligonucleotide can be replaced with guanine and uracil,respectively to form G-U Wobble base pairing with the target mRNA.Sequences containing such replacement moieties are suitable for thecompositions and methods described herein.

The terms “iRNA,” “RNAi agent,” and “RNA interference agent” as usedinterchangeably herein, refer to an agent that contains RNA or modifiedRNA that mediates the targeted cleavage of an RNA transcript via anRNA-induced silencing complex (RISC) pathway. RNAi agents direct thesequence-specific degradation of mRNA through a process known as RNAinterference (RNAi). The iRNA modulates, e.g., inhibits or knocks down,the expression of HNF4α-P2 isoform mRNA in a cell, e.g., a cell within asubject, such as a mammalian subject.

An RNAi agent may be a single stranded RNA or modified RNA thatinteracts with a target RNA sequence, e.g., an HNF4α-P2 isoform targetmRNA sequence, to direct the cleavage of the target RNA. Without wishingto be bound by theory it is believed that long double stranded RNAintroduced into cells is broken down into double stranded shortinterfering RNAs (siRNAs) comprising a sense strand and an antisensestrand by a Type III endonuclease known as Dicer. Dicer, aribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pairshort interfering RNAs with characteristic two base 3′ overhangs. ThesesiRNAs are then incorporated into an RNA-induced silencing complex(RISC) where one or more helicases unwind the siRNA duplex, enabling thecomplementary antisense strand to guide target recognition. Upon bindingto the appropriate target mRNA, one or more endonucleases within theRISC cleave the target to induce silencing. Thus, in one aspect, theinvention relates to a single stranded RNA (ssRNA) (the antisense strandof an siRNA duplex) generated within a cell and which promotes theformation of a RISC complex to effect silencing of the target gene.Accordingly, the term “siRNA” is also used herein to refer to aninterfering RNA (iRNA).

The RNAi agent may be a single-stranded RNA or modified RNA that isintroduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded RNAs are described in U.S. Pat. No. 8,101,348and in Lima et al., (2012) Cell 150:883-894. Any of the RNAi agentsdescribed herein may be used as a single-stranded siRNA as describedherein or as chemically modified by the methods described in Lima etal., (2012) Cell 150:883-894.

An “iRNA” or RNAi agent” for use in the compositions and methodsdescribed herein may be a double stranded RNA and can be referred toherein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to acomplex of ribonucleic acid molecules or modified RNAs, having a duplexstructure comprising two anti-parallel and substantially complementarynucleic acid strands, referred to as having “sense” and “antisense”orientations with respect to a target RNA, e.g., an HNF4α-P2 isoformmRNA.

The majority of nucleotides of each strand of an RNA agent may beribonucleotides, but each or both strands may include one or morenon-ribonucleotides, e.g., a deoxyribonucleotide and/or a modifiednucleotide. In addition, an RNAi agent may include ribonucleotides withchemical modifications at one or more nucleotides. As used herein, theterm “modified nucleotide” refers to a nucleotide having, independently,a modified sugar moiety, a modified inter-nucleotide linkage, and/or amodified nucleobase. Thus, the term “modified nucleotide” encompassessubstitutions, additions or removal of, e.g., a functional group oratom, to inter-nucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the reagents described herein includeany type of modification resulting in a functional RNAi agent, ASO, orother functional oligonucleotide. Any such modifications, as used in ansiRNA-type molecule, are encompassed by “RNAi agent” for the purposes ofthis disclosure. International Patent Application Publication No. WO2016/209862 is incorporated herein by reference in its entirety for thedisclosure of various exemplary RNAi agent compositions andmodifications thereof, and delivery methods and compositions for RNAiagents, useful in the implementation of the present invention.

In an RNAi agent, the duplex region may be of any length that permitsspecific degradation of a desired target RNA through a RISC pathway, andmay range from about 9 to 36 base pairs in length, e.g., about 15-30base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 base pairs in length. Ranges and lengths intermediate tothe above recited ranges and lengths are also contemplated to be part ofthe invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Thehairpin loop may comprise at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least20, at least 23, or more unpaired nucleotides. The hairpin loop may be10 or fewer nucleotides, 8 or fewer unpaired nucleotides, from 4-10unpaired nucleotides, or from 4-8 nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not be, butcan be covalently connected. Where the two strands are connectedcovalently by means other than an uninterrupted chain of nucleotidesbetween the 3′-end of one strand and the 5′-end of the respective otherstrand forming the duplex structure, the connecting structure isreferred to as a “linker.” The RNA strands may have the same or adifferent number of nucleotides. The maximum number of base pairs is thenumber of nucleotides in the shortest strand of the dsRNA minus anyoverhangs that are present in the duplex. In addition to the duplexstructure, an RNAi may comprise one or more nucleotide overhangs.

An RNAi agent may be a dsRNA, each strand of which comprises 19-23nucleotides, that interacts with a target RNA sequence, e.g., anHNF4α-P2 isoform mRNA. Without wishing to be bound by theory, longdouble stranded RNA introduced into cells is broken down into siRNA by aType III endonuclease known as Dicer. Dicer, a ribonuclease-III-likeenzyme, processes the dsRNA into 19-23 base pair short interfering RNAswith characteristic two base 3′ overhangs. The siRNAs are thenincorporated into an RNA-induced silencing complex (RISC) where one ormore helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition. Upon binding to theappropriate target mRNA, one or more endonucleases within the RISCcleave the target to induce silencing. An RNAi agent may be a dsRNA of24-30 nucleotides that interacts with a target RNA sequence to directthe cleavage of the target RNA. A dsRNA may comprise an overhang of atleast one nucleotide; alternatively the overhang can comprise at leasttwo nucleotides, at least three nucleotides, at least four nucleotides,at least five nucleotides, or more. A nucleotide overhang can compriseor consist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.Alternatively, both ends of a dsRNA may be blunt at both ends(blunt-ended), with no nucleotide overhang at either end of themolecule. Most often such a molecule will be double stranded over itsentire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., an HNF4α-P2 isoform mRNA. Asused herein, the term “region of complementarity” refers to the regionon the antisense strand that is substantially complementary to asequence, for example, a target sequence, e.g., an HNF4α-P2 isoform mRNAsequence, as described herein. Where the region of complementarity isnot fully complementary to the target sequence, the mismatches can be inthe internal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA. The term“sense strand” or “passenger strand” as used herein, refers to thestrand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3, or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing. The terms “complementary,” “fullycomplementary” and “substantially complementary” herein can be used withrespect to the base matching between the sense strand and the antisensestrand of a dsRNA, or between the antisense strand of an RNAi agent anda target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an HNF4α-P2 isoform mRNA). For example, a polynucleotideis complementary to at least a part of an HNF4α-P2 isoform mRNA that isnot preset in HNF4α-P1 isoform mRNA if the sequence is substantiallycomplementary to a non-interrupted portion of an HNF4α-P2 isoform mRNA,e.g., bases 1-63 of SEQ ID NO: 1.

The antisense strand polynucleotides disclosed herein may be fullycomplementary to the target HNF4α-P2 isoform mRNA sequence. Theantisense strand polynucleotides disclosed herein may be substantiallycomplementary to the target HNF4α-P2 isoform mRNA sequence and comprisea contiguous nucleotide sequence which is at least about 80%complementary over its entire length to the equivalent region of bases1-53 or bases 1-63 of the nucleotide sequence of SEQ ID NO: 1, or afragment thereof, such as about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, or about 99% complementary.

The antisense polynucleotides disclosed herein may substantiallycomplementary to the target HNF4α-P2 isoform mRNA sequence and comprisea contiguous nucleotide sequence which is at least about 80%complementary over its entire length to any one of the sense strandnucleotide sequences above or in FIG. 7A, or a fragment of any one ofthe sense strand nucleotide sequences above or in FIG. 7A, such as atleast 85%, 90%, 95% complementary, or 100% complementary.

An RNAi agent may include a sense strand that is substantiallycomplementary to an antisense polynucleotide which, in turn, issubstantially or fully complementary to a target HNF4α-P2 isoform mRNAsequence, and wherein the sense strand polynucleotide comprises acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to the equivalent region of a sense nucleotidesequence provided above or in FIG. 7A, or a fragment of a sensenucleotide sequence provided above or in FIG. 7A or 7B, such as about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, or about 99% complementary.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing”, “knockingdown”, and other similar terms, and includes any level of inhibition.The phrase “inhibiting expression of an HNF4α-P2 isoform mRNA,” as usedherein, includes inhibition of expression of any HNF4α gene (such as,e.g., a mouse HNF4α gene, a rat HNF4α gene, a monkey HNF4α gene, or ahuman HNF4α gene) as well as variants or mutants of an HNF4α gene thatencode an HNF4α protein, in its production of HNF4α-P2 isoform mRNA,affecting the stability of HNF4α-P2 isoform mRNA, such as by antisenseor RNAi technologies, or inhibiting translation of HNF4α-P2 isoformmRNA. By “inhibiting expression of an HNF4α-P2 isoform mRNA”, expressionof, stability of, translation of, or activity of HNF4α-P1 isoform mRNAand/or the HNF4α-P1 isoform protein product is not affected to anclinically-relevant or clinically-significant level.

“Inhibiting expression of an HNF4α-P2 isoform mRNA” includes any levelof inhibition of an HNF4α-P2 isoform mRNA, e.g., at least partialsuppression of the expression of an HNF4α-P2 isoform mRNA, such as aninhibition by at least about 20%. Inhibition may be by at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99%.

The expression of an HNF4α-P2 isoform mRNA may be assessed based on thelevel of any variable associated with HNF4α-P2 isoform mRNA expression,e.g., HNF4α-P2 isoform mRNA level or HNF4α-P2 isoform protein level. Theexpression of an HNF4α-P2 isoform mRNA may also be assessed indirectlybased on, e.g., expression of genes and gene products controlled by theHNF4α P1 or P2 gene product(s), or by assay of physiological markersassociated with over-expression of the HNF4α-P2 isoform mRNA, or normalactivity of the HNF4α-P1 isoform mRNA in a patient.

At least partial suppression of the expression of an HNF4α-P2 isoformmRNA, may be assessed by a reduction of the amount of HNF4α-P2 isoformmRNA which can be isolated from or detected in a first cell or group ofcells, e.g., liver cells or tissue, in which an HNF4α gene istranscribed and which has or have been treated such that the expressionof an HNF4α-P2 isoform mRNA is inhibited, as compared to a second cellor group of cells substantially identical to the first cell or group ofcells but which has or have not been so treated (control cells). Thedegree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) \cdot \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}{\%.}$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, asused herein, includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell in vitrowith the RNAi agent, or contacting a cell in vivo with the RNAi agent.The contacting may be done directly or indirectly. Thus, for example,the RNAi agent may be put into physical contact with the cell by theindividual performing the method, or alternatively, the RNAi agent maybe put into a situation that will permit or cause it to subsequentlycome into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, e.g.,the bloodstream or the subcutaneous space, such that the agent willsubsequently reach the tissue where the cell to be contacted is located.For example, the RNAi agent may contain and/or be coupled to a ligand,e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g.,the liver. Combinations of in vitro and in vivo methods of contactingare also possible. For example, a cell may also be contacted in vitrowith an RNAi agent and subsequently transplanted into a subject.

Contacting a cell with an RNAi agent may include “introducing” or“delivering” the RNAi agent into the cell by facilitating or effectinguptake or absorption into the cell. Absorption or uptake of an RNAiagent can occur through unaided diffusive or active cellular processes,or by auxiliary agents or devices. Introducing an RNAi agent into a cellmay be in vitro and/or in vivo. For example, for in vivo introduction,RNAi agent can be injected into a tissue site or administeredsystemically. In vivo delivery can also be done by a beta-glucandelivery system, such as those described in U.S. Pat. Nos. 5,032,401 and5,607,677, and U.S. Patent Application Publication No. 2005/0281781, thetechnical disclosure of which are hereby incorporated herein byreference. In vitro introduction into a cell includes methods known inthe art such as electroporation and lipofection. Further approaches aredescribed herein below and/or are known in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA istranscribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the technical disclosureof which are hereby incorporated herein by reference.

As above, a “patient” or “subject” is an animal, such as a mammal,including a primate (such as a human, a non-human primate, e.g., amonkey and a chimpanzee), a non-primate (such as a cow, a pig, a camel,a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, acat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., aduck or a goose). It is understood that the sequence of the HNF4α-P2isoform mRNA must be sufficiently complementary to the antisense strandof the RNAi agent for the agent to be used in the indicated species.

The patient may be a human, such as a human being treated or assessedfor a disease, disorder or condition that would benefit from reductionin HNF4α-P2 isoform mRNA expression; a human at risk for a disease,disorder or condition that would benefit from reduction in HNF4α-P2isoform mRNA expression; a human having a disease, disorder or conditionthat would benefit from reduction in HNF4α-P2 isoform mRNA expression;and/or human being treated for a disease, disorder or condition thatwould benefit from reduction in HNF4α-P2 isoform mRNA expression asdescribed herein.

By “lower” in the context of a disease marker or symptom is meant aclinically-relevant and/or a statistically significant decrease in suchlevel. The decrease can be, for example, at least 10%, at least 20%, atleast 30%, at least 40%, or more, down to a level accepted as within therange of normal for an individual without such disorder, or to below thelevel of detection of the assay. The decrease may be down to a levelaccepted as within the range of normal for an individual without suchdisorder which can also be referred to as a normalization of a level.For example, lowering bilirubin to within a range of from 0.3-1 mg/dL(5.1-17 micromol/L) would be considered to be within the range of normalfor a subject. The reduction may be the normalization of the level of asign or symptom of a disease, a reduction in the difference between thesubject level of a sign of the disease and the normal level of the signfor the disease (e.g., to the upper level of normal when the value forthe subject must be decreased to reach a normal value, and to the lowerlevel of normal when the value for the subject must be increased toreach a normal level). The methods may include a clinically relevantinhibition of expression of an HNF4α-P2 isoform mRNA, e.g., asdemonstrated by a clinically relevant outcome after treatment of asubject with an agent to reduce the expression of an HNF4α-P2 isoformmRNA.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of an HNF4α-P2 isoform mRNA, refers to areduction in the likelihood that a subject will develop a symptomassociated with such disease, disorder, or condition, e.g., liverdisease, liver damage, liver inflammation, or liver failure, such asacute liver failure, ALD, AH, or ACLF. The likelihood of developingliver disease, liver damage, liver inflammation, or liver failure, suchas acute liver failure, ALD, AH, or ACLF is reduced, for example, whenan individual having one or more risk factors for liver disease, liverdamage, liver inflammation, or liver failure, such as acute liverfailure, ALD, AH, or ACLF, e.g., a genetic disorder, either fails todevelop liver disease, liver damage, liver inflammation, or liverfailure, such as acute liver failure, ALD, AH, or ACLF, or signs orsymptoms thereof, or develops liver disease, liver damage, liverinflammation, or liver failure, such as acute liver failure, ALD, AH, orACLF, or signs or symptoms thereof, with less severity relative to apopulation having the same risk factors and not receiving treatment asdescribed herein. The failure to develop a disease, disorder orcondition, or the reduction in the development of a symptom associatedwith such a disease, disorder or condition (e.g., by at least about 10%on a clinically accepted scale for that disease or disorder), or theexhibition of delayed symptoms (e.g., by days, weeks, months, or years)is considered effective prevention. Prevention can requireadministration of more than one dose of an agent described herein.

As used herein, a “disease or disorder that would benefit from reductionin HNF4α-P2 isoform mRNA levels” may be a disease or disorder associatedwith liver disease, liver damage, or liver failure. For example, thisterm includes any disorder, disease or condition resulting in one ormore signs or symptoms of acute liver failure, ALD, AH, or ACLF.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving liver disease, liver damage, liver inflammation, or liverfailure, such as acute liver failure, ALD, AH, or ACLF, is sufficient toeffect treatment of the disease (e.g., by diminishing, ameliorating ormaintaining the existing disease or one or more symptoms of disease).The “therapeutically effective amount” may vary depending on thesequence and chemical composition of the RNAi agent, how the agent isadministered, the disease and its severity, the patient's history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe subject to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving liver disease, liver damage, liver inflammation, or liverfailure, such as acute liver failure, ALD, AH, or ACLF, is sufficient toprevent or ameliorate the disease or one or more symptoms of thedisease. Ameliorating the disease includes slowing the course of thedisease or reducing the severity of later-developing disease. The“prophylactically effective amount” may vary depending on the RNAiagent, how the agent is administered, the disease and its severity, thepatient's history, age, weight, family history, genetic makeup, thetypes of preceding or concomitant treatments, if any, and otherindividual characteristics of the subject to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. RNAi agents employed in the methodsdescribed herein may be administered in a sufficient amount to produce areasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. Samples may be derived from theliver (e.g., whole liver or certain segments of liver or certain typesof cells in the liver, such as, e.g., hepatocytes). A “sample derivedfrom a subject” may refer to blood drawn from the subject or plasmaisolated therefrom, saliva, or urine, typically a 24 hour urine sample.

Described herein are RNAi agents that inhibit the expression of anHNF4α-P2 isoform mRNA. The RNAi agent may include double strandedribonucleic acid (dsRNA) molecules for inhibiting the expression of anHNF4α-P2 isoform mRNA in a cell, such as a cell within a subject, e.g.,a mammal, such as a human having liver disease, liver damage, liverinflammation, or liver failure, such as acute liver failure, ALD, AH, orACLF. The dsRNA includes an antisense strand having a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of an HNF4α-P2 isoform mRNA. The region ofcomplementarity is about 30 nucleotides or less in length (e.g., about30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides orless in length). Upon contact with a cell expressing the HNF4α-P2isoform mRNA, the iRNA inhibits the expression of the HNF4α-P2 isoformmRNA (e.g., a human, a primate, a non-primate, or a bird HNF4α-P2isoform mRNA) by at least about 10% as assayed by, for example, aPCR-based method, or by a protein-based method, such as byimmunofluorescence analysis, using, for example, western blotting, orflowcytometric techniques. Inhibition of expression may determined bythe qPCR method provided in the examples. For in vitro assessment ofactivity, percent inhibition is determined using the methods providedherein at a single dose at, for example, a 10 nM duplex finalconcentration. For in vivo studies, the level after treatment can becompared to, for example, an appropriate historical control or a pooledpopulation sample control to determine the level of reduction, e.g.,when a baseline value is no available for the subject.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of an HNF4α-P2isoform mRNA. The other strand (the sense strand) includes a region thatis complementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 nucleotides in length. Ranges and lengths intermediateto the above recited ranges and lengths are also contemplated to be partof the invention.

The dsRNA may be between about 15 and about 23 nucleotides in length, orbetween about 25 and about 30 nucleotides in length. In general, thedsRNA is long enough to serve as a substrate for the Dicer enzyme. Forexample, it is well known in the art that dsRNAs longer than about 21-23nucleotides can serve as substrates for Dicer. As the ordinarily skilledperson will also recognize, the region of an RNA targeted for cleavagewill most often be part of a larger RNA molecule, often an mRNAmolecule. Where relevant, a “part” of an mRNA target is a contiguoussequence of an mRNA target of sufficient length to allow it to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, to the extent that it becomes processed to afunctional duplex, of e.g., 15-30 base pairs, that targets a desired RNAfor cleavage, an RNA molecule or complex of RNA molecules having aduplex region greater than 30 base pairs may be a dsRNA. Thus, anordinarily skilled artisan will recognize that an miRNA is a dsRNA. AdsRNA may not be a naturally occurring miRNA. An RNAi agent useful totarget HNF4α-P2 isoform mRNA expression may not be generated in thetarget cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end, or both ends of either an antisense orsense strand of a dsRNA. Longer, extended overhangs are possible.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch™,Applied Biosystems™, Inc. iRNA compounds may be prepared using atwo-step procedure.

First, the individual strands of the double stranded RNA molecule areprepared separately. Then, the component strands are annealed. Theindividual strands of the siRNA compound can be prepared usingsolution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides can be prepared using solution-phase orsolid-phase organic synthesis or both.

A dsRNA may include at least two nucleotide sequences, a sense sequenceand an anti-sense sequence. The sense strand sequence is selected fromthe group of sequences provided above or in FIG. 7A, and thecorresponding nucleotide sequence of the antisense strand of the sensestrand is selected from the group of sequences provided in above or inFIG. 7A. One of the two sequences may be complementary to the other ofthe two sequences, with one of the sequences being substantiallycomplementary to a sequence of an HNF4α-P2 isoform mRNA. As such, adsRNA may include two oligonucleotides, where one oligonucleotide isdescribed as the sense strand in above or in FIG. 7A, and the secondoligonucleotide is described as the corresponding antisense strand ofthe sense strand in above or in FIG. 7A. The substantially complementarysequences of the dsRNA may be contained on separate oligonucleotides oron a single oligonucleotide.

It will be understood that, although the sequences in FIG. 7A aredescribed as modified and/or conjugated sequences, the RNA of the iRNAdescribed herein, e.g., a dsRNA, may comprise any one of the sequencesset forth in any one of FIG. 7A that is un-modified, un-conjugated,and/or modified and/or conjugated differently than described therein.

A double stranded ribonucleic acid (dsRNA) for inhibiting expression ofHNF4α-P2 isoform mRNA may comprise, consist essentially of, or consistof a sense strand and an antisense strand, wherein the sense strandcomprises the nucleotide sequence of a sense strand (e.g., as in FIG.7A) and the antisense strand comprises the nucleotide sequence of thecorresponding antisense strand (e.g., as in FIG. 7A).

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference. However,others have found that shorter or longer RNA duplex structures can alsobe effective. In the aspects described above, by virtue of the nature ofthe oligonucleotide sequences provided herein, dsRNAs described hereincan include at least one strand of a length of minimally 21 nucleotides.It can be reasonably expected that shorter duplexes minus only a fewnucleotides on one or both ends can be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a sequence of atleast 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derivedfrom one of the sequences provided herein, and differing in theirability to inhibit the expression of an HNF4α-P2 isoform mRNA by notmore than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNAcomprising the full sequence, are contemplated to be within the scope ofthe present disclosure.

In addition, the RNAs described herein identify a site in an HNF4α-P2isoform mRNA that is susceptible to RISC-mediated cleavage. As such, thepresent invention further features iRNAs that target within this site.As used herein, an iRNA is said to target within a particular site of anRNA transcript if the RNAi agent promotes selective cleavage of thetranscript anywhere within that particular site. Such an RNAi agent willgenerally include at least about 15 contiguous nucleotides from one ofthe sequences provided herein coupled to additional nucleotide sequencestaken from the region contiguous to the selected sequence in an HNF4αgene.

While a target sequence is generally about 15-30 nucleotides in length,there can be variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages may provide guidance for the identification of optimaltarget sequences for any given gene target, but an empirical approachmay also be taken in which a “window” or “mask” of a given size (as anon-limiting example, 21 nucleotides) is literally or figuratively(including, e.g., in silico) placed on the target RNA sequence toidentify sequences in the size range that can serve as target sequences.By moving the sequence “window” progressively one nucleotide upstream ordownstream of an initial target sequence location, the next potentialtarget sequence can be identified, until the complete set of possiblesequences is identified for any given target size selected. Thisprocess, coupled with systematic synthesis and testing of the identifiedsequences (using assays as described herein or as known in the art) toidentify those sequences that perform optimally can identify those RNAsequences that, when targeted with an RNAi agent, mediate the bestinhibition of target gene expression. Thus, while the sequencesidentified herein represent effective target sequences, it iscontemplated that further optimization of inhibition efficiency can beachieved by progressively “walking the window” one nucleotide upstreamor downstream of the given sequences to identify sequences with equal orbetter inhibition characteristics.

Further, based on the present disclosure, it is contemplated that forany sequence identified herein, further optimization could be achievedby systematically either adding or removing nucleotides to generatelonger or shorter sequences and testing those sequences generated bywalking a window of the longer or shorter size up or down the target RNAfrom that point. Again, coupling this approach to generating newcandidate targets with testing for effectiveness of iRNAs based on thosetarget sequences in an inhibition assay as known in the art and/or asdescribed herein can lead to further improvements in the efficiency ofinhibition. Further still, such optimized sequences can be adjusted by,e.g., the introduction of modified nucleotides as described herein or asknown in the art, addition or changes in overhang, or othermodifications as known in the art and/or discussed herein to furtheroptimize the molecule (e.g., increasing serum stability or circulatinghalf-life, increasing thermal stability, enhancing transmembranedelivery, targeting to a particular location or cell type, increasinginteraction with silencing pathway enzymes, increasing release fromendosomes) as an expression inhibitor.

An RNAi agent as described herein can contain one or more mismatches tothe target sequence. In one aspect, an RNAi agent as described hereincontains no more than 3 mismatches. If the antisense strand of the RNAiagent contains mismatches to a target sequence, it may be preferablethat the area of mismatch is not located in the center of the region ofcomplementarity. If the antisense strand of the RNAi agent containsmismatches to the target sequence, it may be preferable that themismatch be restricted to be within the last 5 nucleotides from eitherthe 5′- or 3′-end of the region of complementarity. For example, for a23 nucleotide RNAi agent the strand which is complementary to a regionof an HNF4α-P2 isoform mRNA, generally does not contain any mismatchwithin the central 13 nucleotides. The methods described herein ormethods known in the art can be used to determine whether an RNAi agentcontaining a mismatch to a target sequence is effective in inhibitingthe expression of an HNF4α-P2 isoform mRNA. Consideration of theefficacy of iRNAs with mismatches in inhibiting expression of anHNF4α-P2 isoform mRNA is important, especially if the particular regionof complementarity in an HNF4α-P2 isoform mRNA is known to havepolymorphic sequence variation within the population.

The RNA of the RNAi agent described herein, e.g., a dsRNA, may beun-modified, and does not comprise, e.g., chemical modifications and/orconjugations known in the art and described herein. The RNA of an RNAiagent described herein, e.g., a dsRNA, may be chemically modified toenhance stability or other beneficial characteristics. Substantially allof the nucleotides of an RNAi agent may be modified. All of thenucleotides of an RNAi agent may be modified. RNAi agents in which“substantially all of the nucleotides are modified” are largely but notwholly modified and can include not more than 5, 4, 3, 2, or 1unmodified nucleotides.

The nucleic acids described herein may be synthesized and/or modified bymethods well established in the art. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA agents useful in themethods described herein include, but are not limited to RNAs containingmodified backbones or no natural internucleoside linkages. RNAs havingmodified backbones include, among others, those that do not have aphosphorus atom in the backbone. Modified RNAs that do not have aphosphorus atom in their internucleoside backbone are also be consideredto be oligonucleosides.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and RE39464, thetechnical disclosure of each of which are hereby incorporated herein byreference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and C component parts.

Suitable RNA mimetics in RNAi agents in which both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units maybe replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an RNA mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA), which includes chiral γ-PNAs. In PNA compounds, the sugarbackbone of an RNA is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative U.S. patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, the technical disclosure ofeach of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs are described in, forexample, in Nielsen et al., Science, 1991, 254, 1497-1500. Additionalnucleic acid modifications are broadly-known.

An iRNA may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds featured in the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C., and areexemplary base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

The RNA of an RNAi agent can also be modified to include one or morelocked nucleic acids (LNA). A locked nucleic acid is a nucleotide havinga modified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects.

The RNAi agent may comprise one or more monomers that are UNA (unlockednucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, whereinany of the bonds of the sugar has been removed, forming an unlocked“sugar” residue. In one example, UNA also encompasses monomer with bondsbetween C1′-C4′ have been removed (i.e., the covalentcarbon-oxygen-carbon bond between the C1′ and C4′ carbons). In anotherexample, the C2′-C3′ bond (i.e., the covalent carbon-carbon bond betweenthe C2′ and C3′ carbons) of the sugar has been removed). RepresentativeU.S. publications that teach the preparation of UNA include, but are notlimited to, U.S. Pat. No. 8,314,227; and US Patent ApplicationPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, thetechnical disclosure of each of which are hereby incorporated herein byreference.

An RNAi agent may also include one or more “conformationally restrictednucleotides” (“CRN”). CRN are nucleotide analogs with a linkerconnecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbonsof ribose. CRN lock the ribose ring into a stable conformation andincrease the hybridization affinity to mRNA. The linker is of sufficientlength to place the oxygen in an optimal position for stability andaffinity resulting in less ribose ring puckering. Representativepublications that teach the preparation of certain of the above notedCRN include, but are not limited to, US Patent Application PublicationNo. 2013/0190383; and PCT Publication No. WO 2013/036868, the technicaldisclosure of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-0-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

Other modifications of the nucleotides of an iRNA include a 5′ phosphateor 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimicon the antisense strand of an RNAi agent. Suitable phosphate mimics aredisclosed in, for example US Patent Application Publication No.2012/0157511, the technical disclosure of which are incorporated hereinby reference.

Another possible modification of an iRNA involves chemically linking tothe RNA one or more ligands, moieties or conjugates that enhance theactivity, cellular distribution or cellular uptake of the iRNA. Suchmoieties include but are not limited to lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., beryl-S-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine orhexylamino-carbonyloxycholesterol moiety.

A ligand may alter the distribution, targeting or lifetime of an RNAiagent into which it is incorporated. A ligand may provide an enhancedaffinity for a selected target, e.g., molecule, cell or cell type,compartment, e.g., a cellular or organ compartment, tissue, organ orregion of the body, as, e.g., compared to a species absent such aligand. It may be preferable that ligands will not take part in duplexpairing in a duplexed nucleic acid. Ligands can include a naturallyoccurring substance, such as a protein (e.g., human serum albumin (HSA),low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin,N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands may also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, mucin carbohydrate, multivalentlactose, monovalent or multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate,polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate,vitamin B 12, vitamin A, biotin, or an RGD peptide or RGD peptidemimetic. Ligands may include monovalent or multivalent galactose.Ligands may include cholesterol.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine), and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g.,biotin), transport/absorption facilitators (e.g., aspirin, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands may be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They mayalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand may be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-KB.

The ligand may be a substance, e.g., a drug, which can increase theuptake of the RNAi agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

A ligand attached to an iRNA may act as a pharmacokinetic modulator (PKmodulator). PK modulators include lipophiles, bile acids, steroids,phospholipid analogues, peptides, protein binding agents, PEG, vitaminsetc. Exemplary PK modulators include, but are not limited to,cholesterol, fatty acids, cholic acid, lithocholic acid,dialkylglycerides, diacylglyceride, phospholipids, sphingolipids,naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides thatcomprise a number of phosphorothioate linkages are also known to bind toserum protein, thus short oligonucleotides, e.g., oligonucleotides ofabout 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple ofphosphorothioate linkages in the backbone are also amenable to thepresent invention as ligands (e.g., as PK modulating ligands). Inaddition, aptamers that bind serum components (e.g., serum proteins) arealso suitable for use as PK modulating ligands.

The ligand may be a cell-permeation agent, such as a helicalcell-permeation agent. The agent may be amphipathic. An exemplary agentis a peptide such as that or antennopedia. If the agent is a peptide, itcan be modified, including a peptidylmimetic, invertomers, non-peptideor pseudo-peptide linkages, and use of D-amino acids. The helical agentmay be an alpha-helical agent, which may have a lipophilic and alipophobic phase.

The ligand may be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to RNAi agentscan affect pharmacokinetic distribution of the RNAi agent, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety may be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic may be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). RFGF. The peptide moiety can be a“delivery” peptide, which can carry large polar molecules includingpeptides, oligonucleotides, and protein across cell membranes. Forexample, sequences from the HIV Tat protein) and the DrosophilaAntennapedia protein (have been found to be capable of functioning asdelivery peptides. A peptide or peptidomimetic can be encoded by arandom sequence of DNA, such as a peptide identified from aphage-display library, or one-bead-one-compound (OBOC) combinatoriallibrary. Examples of a peptide or peptidomimetic tethered to a dsRNAagent via an incorporated monomer unit for cell targeting purposes is anarginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptidemoiety can range in length from about 5 amino acids to about 40 aminoacids. The peptide moieties can have a structural modification, such asto increase stability or direct conformational properties. Any of thestructural modifications described below can be utilized.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, a a-helical linear peptide (e.g., LL-37 or Ceropin PI), adisulfide bond-containing peptide (e.g., a-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen.

Ligand-conjugated oligonucleotides may be synthesized by the use of anoligonucleotide that bears a pendant reactive functionality, such asthat derived from the attachment of a linking molecule onto theoligonucleotide. This reactive oligonucleotide may be reacted directlywith commercially-available ligands, ligands that are synthesizedbearing any of a variety of protecting groups, or ligands that have alinking moiety attached thereto.

The RNAi agents may be conveniently and routinely made through thewell-known technique of solid-phase synthesis. Equipment for suchsynthesis is sold by several vendors including, for example, AppliedBiosystems™ (Foster City, Calif.). Any other means for such synthesisknown in the art may additionally or alternatively be employed. It isalso known to use similar techniques to prepare other oligonucleotides,such as the phosphorothioates and alkylated derivatives. Inligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks. When using nucleotide-conjugate precursors that alreadybear a linking moiety, the synthesis of an oligonucleotide comprising alinker may be first completed, and the ligand molecule may then bereacted with the linking moiety to form the ligand-conjugatedoligonucleotide. Oligonucleotides or linked nucleosides may besynthesized by an automated synthesizer using phosphoramidites derivedfrom ligand-nucleoside conjugates in addition to the standardphosphoramidites and non-standard phosphoramidites that are commerciallyavailable and routinely used in oligonucleotide synthesis.

An iRNA agent may further comprises a carbohydrate.Carbohydrate-conjugated RNAi agent are advantageous for the in vivodelivery of nucleic acids, as well as compositions suitable for in vivotherapeutic use, as described herein. As used herein, “carbohydrate”refers to a compound which is either a carbohydrate per se made up ofone or more monosaccharide units having at least 6 carbon atoms (whichcan be linear, branched or cyclic) with an oxygen, nitrogen, or sulfuratom bonded to each carbon atom; or a compound having as a part thereofa carbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8). A carbohydrate conjugate may be or may comprise amonosaccharide.

One or more GalNAc (N-Acetylgalactosamine) or GalNAc derivative moietiesmay be attached to an RNAi agent by a linker, such as a monovalent,bivalent, or trivalent linker. An example of a GalNAc-containing moietyis L96 (N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol(Hyp-(GalNAc-alkyl)₃). Additional carbohydrate conjugates (and linkers)suitable for use include those described in PCT Publication Nos. WO2014/179620, WO 2014/179627, and WO 2016/209862, each of which isincorporated herein by reference. An RNAi agent may comprise anoligonucleotide conjugated to a bivalent or trivalent branched linkerselected from the structures of formula (XXIV)-(XXXV) depicted inInternational Patent Application Publication No. WO 2016/209862.

The conjugate or ligand described herein can be attached to an iRNAoligonucleotide with a linker that can be cleavable or non-cleavable.The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen, nitrogen, or sulfur, a unit such as NH, C(O), C(O)NH, SO, SO₂,SO₂NH, or a chain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(H), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. The linker may consist of between about 1-24atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16,7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. The cleavable linkinggroup may be cleaved at least about 10 times, 20, times, 30 times, 40times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 timesfaster in a target cell or under a first reference condition (which,e.g., may be selected to mimic or represent intracellular conditions)than in the blood of a subject, or under a second reference condition(which can, e.g., be selected to mimic or represent conditions found inthe blood or serum). Cleavable linking groups are susceptible tocleavage agents, e.g., pH, redox potential or the presence ofdegradative molecules. Generally, cleavage agents are more prevalent orfound at higher levels or activities inside cells than in serum orblood. Examples of such degradative agents include: redox agents whichare selected for particular substrates or which have no substratespecificity, including, e.g., oxidative or reductive enzymes orreductive agents such as mercaptans, present in cells, that can degradea redox cleavable linking group by reduction; esterases; endosomes oragents that can create an acidic environment, e.g., those that result ina pH of five or lower; enzymes that can hydrolyze or degrade an acidcleavable linking group by acting as a general acid, peptidases (whichcan be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond may be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker may include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds may be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes. In general,the suitability of a candidate cleavable linking group can be evaluatedby testing the ability of a degradative agent (or condition) to cleavethe candidate linking group. It will also be desirable to also test thecandidate cleavable linking group for the ability to resist cleavage inthe blood or when in contact with other non-target tissue. Thus, one candetermine the relative susceptibility to cleavage between a first and asecond condition, where the first is selected to be indicative ofcleavage in a target cell and the second is selected to be indicative ofcleavage in other tissues or biological fluids, e.g., blood or serum.The evaluations can be carried out in cell free systems, in cells, incell culture, in organ or tissue culture, or in whole animals. It can beuseful to make initial evaluations in cell-free or culture conditionsand to confirm by further evaluations in whole animals.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The iRNA agents may be chimericcompounds. “Chimeric” iRNA compounds or “chimeras,” are iRNA agents,e.g., dsRNAs, which contain two or more chemically distinct regions,each made up of at least one monomer unit, e.g., a nucleotide in thecase of a dsRNA compound. These RNAi agents typically contain at leastone region wherein the RNA is modified so as to confer upon the RNAiagent increased resistance to nuclease degradation, increased cellularuptake, and/or increased binding affinity for the target nucleic acid.An additional region of the RNAi agent can serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofiRNA inhibition of gene expression. Consequently, comparable results canoften be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

The RNA of an RNAi agent may be modified by a non-ligand group.Non-ligand molecules may be conjugated to RNAi agents in order toenhance the activity, cellular distribution or cellular uptake of theiRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties include lipidmoieties, such as cholesterol, cholic acid, a thioether, e.g.,hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Typical conjugation protocols involve the synthesis of an RNA bearing anamino linker at one or more positions of the sequence. The amino groupis then reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction can beperformed either with the RNA still bound to a solid support orfollowing cleavage of the RNA, in solution phase. Purification of theRNA conjugate by HPLC typically affords the pure conjugate.

The delivery of an RNAi agent to a cell e.g., a cell within a subject,such as a human subject (e.g., a subject in need thereof) can beachieved in a number of different ways. For example, delivery may beperformed by contacting a cell with an RNAi agent either in vitro or invivo. In vivo delivery may also be performed directly by administering acomposition comprising an RNAi agent, e.g., a dsRNA, to a subject.Alternatively, in vivo delivery may be performed indirectly byadministering one or more vectors that encode and direct the expressionof the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an RNAi agent (see e.g., WO94/02595, which is incorporated herein by reference). For in vivodelivery, factors to consider in order to deliver an iRNA moleculeinclude, for example, biological stability of the delivered molecule,prevention of non-specific effects, and accumulation of the deliveredmolecule in the target tissue. The non-specific effects of an RNAi agentcan be minimized by local administration, for example, by directinjection or implantation into a tissue or topically administering thepreparation. Local administration to a treatment site maximizes localconcentration of the agent, limits the exposure of the agent to systemictissues that can otherwise be harmed by the agent or that can degradethe agent, and permits a lower total dose of the RNAi agent to beadministered. Local administration of an RNAi agent has resulted insuccessful knockdown of gene products. For example, intraocular deliveryof a VEGF dsRNA by intravitreal injection in cynomolgus monkeys andsubretinal injections in mice were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume and can prolong survival of tumor-bearingmice. RNA interference has also shown success with local delivery to theCNS by direct injection, and to the lungs by intranasal administration.

For administering an RNAi agent systemically for the treatment of adisease, the RNA can be modified or alternatively delivered using a drugdelivery system; both methods act to prevent the rapid degradation ofthe dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA orthe pharmaceutical carrier can also permit targeting of the iRNAcomposition to the target tissue and avoid undesirable off-targeteffects. RNAi agnets can be modified by chemical conjugation tolipophilic groups such as cholesterol to enhance cellular uptake andprevent degradation. For example, an RNAi agent directed against ApoBconjugated to a lipophilic cholesterol moiety was injected systemicallyinto mice and resulted in knockdown of apoB mRNA in both the liver andjejunum. Conjugation of an RNAi agent to an aptamer has been shown toinhibit tumor growth and mediate tumor regression in a mouse model ofprostate cancer. Alternatively, the RNAi agent may be delivered usingdrug delivery systems such as a nanoparticle, a dendrimer, a polymer,liposomes, or a cationic delivery system.

Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an RNAiagent by the cell. Cationic lipids, dendrimers, or polymers can eitherbe bound to an iRNA, or induced to form a vesicle or micelle thatencases an RNAi agent. The formation of vesicles or micelles furtherprevents degradation of the RNAi agent when administered systemically.Methods for making and administering cationic-iRNA complexes are wellwithin the abilities of one skilled in the art. Some non-limitingexamples of drug delivery systems useful for systemic delivery of RNAiagents include DOTAP, Oligofectamine, “solid nucleic acid lipidparticles”, cardiolipin, polyethyleneimine, and polyamidoamines. An RNAiagent may form a complex with cyclodextrin for systemic administration.Methods for administration and pharmaceutical compositions of RNAiagents and cyclodextrins are described, for example, in U.S. Pat. No.7,427,605.

An RNAi agent targeting the HNF4α-P2 isoform mRNA can be expressed fromgenes inserted into DNA or RNA vectors. Expression can be transient (onthe order of hours to weeks) or sustained (weeks to months or longer),depending upon the specific construct used and the target tissue or celltype. These transgenes can be introduced as a linear construct, acircular plasmid, or a viral vector, which can be an integrating ornon-integrating vector. The transgene can also be constructed to permitit to be inherited as an extrachromosomal plasmid. The individual strandor strands of an iRNA can be transcribed from a promoter on anexpression vector. Where two separate strands are to be expressed togenerate, for example, a dsRNA, two separate expression vectors can beco-introduced (e.g., by transfection or infection) into a target cell.Alternatively, each individual strand of a dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. AdsRNA may be expressed as inverted repeat polynucleotides joined by alinker polynucleotide sequence such that the dsRNA has a stem and loopstructure.

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA-expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to: (a)adenovirus vectors; (b) retrovirus vectors, including, but not limitedto, lentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e)SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors;(h) picornavirus vectors; (i) pox virus vectors such as an orthopox,e.g., vaccinia virus vectors or avipox, e.g., canary pox or fowl pox;and (j) a helper-dependent or gutless adenovirus. Replication-defectiveviruses can also be advantageous. Different vectors will or will notbecome incorporated into the cells' genome. The constructs can includeviral sequences for transfection, if desired. Alternatively, theconstruct can be incorporated into vectors capable of episomalreplication, e.g., EPV and EBV vectors. Constructs for the recombinantexpression of an iRNA will generally require regulatory elements, e.g.,promoters, enhancers, etc., to ensure the expression of the iRNA intarget cells. Other factors to consider for vectors and constructs areknown in the art.

Also provided herein are pharmaceutical compositions and formulationswhich include the RNAi agents. In one aspect, provided herein arepharmaceutical compositions containing an RNAi agent, as describedherein, and a pharmaceutically acceptable carrier. The pharmaceuticalcompositions containing the RNAi agent are useful for treating a diseaseor disorder associated with the expression or activity of an HNF4α-P2isoform mRNA, such as, liver disease, liver damage, liver inflammation,or liver failure, such as acute liver failure, ALD, AH, or ACLF.

Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by intravenous (IV) or forsubcutaneous delivery. Another example is compositions that areformulated for direct delivery into the liver, e.g., by infusion intothe liver, such as by continuous pump infusion.

The pharmaceutical compositions may be formulated and/or administered indosages sufficient to inhibit expression of an HNF4α-P2 isoform mRNA. Ingeneral, a suitable dose of an RNAi agent will be in the range of fromabout 0.001 to about 200.0 milligrams per kilogram body weight of therecipient per day, generally in the range of from about 1 to 50 mg perkilogram body weight per day. A suitable dose of an RNAi agent may be inthe range of from about 0.1 mg/kg to about 5.0 mg/kg, e.g., about 0.3mg/kg and about 3.0 mg/kg. A repeat-dose regimen may includeadministration of a therapeutic amount of RNAi agent on a regular basis,such as every other day or once a year. The RNAi agent may beadministered from about once per month to about once per quarter (e.g.,about once every three months). After an initial treatment regimen, thetreatments may be administered on a less frequent basis.

The methods herein may include administering a composition featuredherein such that expression of the target HNF4α-P2 isoform mRNA isdecreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24hours, 28, 32, or about 36 hours. Expression of the target HNF4α-P2isoform mRNA may be decreased for an extended duration, e.g., at leastabout two, three, four days or more, e.g., about one week, two weeks,three weeks, or four weeks or longer.

Before administration of a full dose of the RNAi agent, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immune-stimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject,including, but not limited to, the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual RNAi agents encompassed herein canbe made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein lies generally within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods described herein, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose can be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma can be measured, for example, by high performance liquidchromatography.

The pharmaceutical composition may be administered in a number of waysdepending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration can be topical (e.g., by atransdermal patch), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal, oral or parenteral. Parenteral administrationincludes intravenous, intraarterial, subcutaneous, intraperitoneal orintramuscular injection or infusion; subdermal, e.g., via an implanteddevice; or intracranial, e.g., by intraparenchymal, intrathecal orintraventricular, administration. The RNAi agent may be delivered in amanner to target a particular tissue, such as the liver (e.g., thehepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids, and powders. Conventionalpharmaceutical carriers, aqueous, powder, or oily bases, thickeners andthe like can be necessary or desirable. Suitable topical formulationsinclude those in which the RNAi agents featured herein are in admixturewith a topical delivery agent such as lipids, liposomes, fatty acids,fatty acid esters, steroids, chelating agents, and surfactants. Suitablelipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). RNAi agents featuredherein can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, RNAi agentsmay be complexed with lipids, such as cationic lipids. Suitable fattyacids and esters include, but are not limited to, arachidonic acid,oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, anacylcholine, or a C₁₋₂o alkyl ester (e.g., isopropylmyristate IPM),monoglyceride or diglyceride; or pharmaceutically acceptable saltthereof. Topical formulations are described in detail in U.S. Pat. No.6,747,014.

An RNAi agent for use in the methods described herein can be formulatedfor delivery in a membranous molecular assembly, e.g., a liposome or amicelle. As used herein, the term “liposome” refers to a vesiclecomposed of amphiphilic lipids arranged in at least one bilayer, e.g.,one bilayer or a plurality of bilayers. Liposomes include unilamellarand multilamellar vesicles that have a membrane formed from a lipophilicmaterial and an aqueous interior. The aqueous portion contains the RNAiagent composition. The lipophilic material isolates the aqueous interiorfrom an aqueous exterior, which typically does not include the RNAiagent, although in some examples, it may. Liposomes are useful for thetransfer and delivery of active ingredients to the site of action.Because the liposomal membrane is structurally similar to biologicalmembranes, when liposomes are applied to a tissue, the liposomal bilayerfuses with bilayer of the cellular membranes. As the merging of theliposome and cell progresses, the internal aqueous contents that includethe RNAi agent are delivered into the cell where the RNAi agent canspecifically bind to a target RNA and can mediate RNAi agent. In somecases, the liposomes are also specifically targeted, e.g., to direct theRNAi agent to particular cell types.

A liposome containing a RNAi agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAiagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the RNAi agentand condense around the RNAi agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of RNAi agent.

If necessary, a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the technical disclosure of which is incorporated herein byreference. Liposome formation also may prepared or used as described inU.S. Pat. Nos. 4,897,355 or 5,171,678. Commonly used techniques forpreparing lipid aggregates of appropriate size for use as deliveryvehicles include sonication and freeze-thaw plus extrusion.Microfluidization can be used when consistently small (50 to 200 nm) andrelatively uniform aggregates are desired. These methods are readilyadapted to packaging RNAi agent preparations into liposomes.

For delivery of the RNAi agent in a spray, a suitable formulation can beput into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray. Propellants may include hydrogen-containingchlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether,and diethyl ether. HFA 134a (1, 1,1,2 tetrafluoroethane) may be used.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions, or solutionsin water or nonaqueous media, capsules, gel capsules, sachets, tablets,or minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids, or binders can be desirable. Oral formulations may bethose in which an RNAi agent is administered in conjunction with one ormore penetration enhancer surfactants and chelators. Suitablesurfactants include fatty acids and/or esters or salts thereof, bileacids and/or salts thereof. Suitable bile acids/salts includechenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA),cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid,glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodiumglycodihydrofusidate. Suitable fatty acids include arachidonic acid,undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, anacylcholine, or a monoglyceride or a diglyceride; or a pharmaceuticallyacceptable salt thereof (e.g., sodium). Combinations of penetrationenhancers may be used, for example, fatty acids/salts in combinationwith bile acids/salts. One exemplary combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. RNAiagents may be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. DsRNA complexingagents include poly-amino acids; polyimines; polyacrylates;polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationizedgelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) andstarches; polyalkylcyanoacrylates; DEAE-derivatized polyimines,pollulans, celluloses and starches. Suitable complexing agents includechitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, poly spermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), poly amino styrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Patent Application Publication No. 2003/0027780, andU.S. Pat. No. 6,747,014.

The compositions can be prepared and formulated as emulsions. Emulsionsare typically heterogeneous systems of one liquid dispersed in anotherin the form of droplets usually exceeding 0.1 μm in diameter. Emulsionsare often biphasic systems comprising two immiscible liquid phasesintimately mixed and dispersed with each other. In general, emulsionscan be of either the water-in-oil (w/o) or the oil-in-water (o/w)variety. When an aqueous phase is finely divided into and dispersed asminute droplets into a bulk oily phase, the resulting composition iscalled a water-in-oil (w/o) emulsion. Alternatively, when an oily phaseis finely divided into and dispersed as minute droplets into a bulkaqueous phase, the resulting composition is called an oil-in-water (o/w)emulsion. Emulsions can contain additional components in addition to thedispersed phases, and the active drug which can be present as a solutionin either aqueous phase, oily phase or itself as a separate phase.Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, andanti-oxidants can also be present in emulsions as needed. Pharmaceuticalemulsions can also be multiple emulsions that are comprised of more thantwo phases such as, for example, in the case of oil-in-water-in-oil(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complexformulations often provide certain advantages that simple binaryemulsions do not. Multiple emulsions in which individual oil droplets ofan o/w emulsion enclose small water droplets constitute a w/o/wemulsion. Likewise, a system of oil droplets enclosed in globules ofwater stabilized in an oily continuous phase provides an o/w/o emulsion.

An RNAi agent may be incorporated into a particle, e.g., amicroparticle. Microparticles can be produced by spray-drying but mayalso be produced by other methods including lyophilization, evaporation,fluid bed drying, vacuum drying, or a combination of these techniques.

Penetration enhancers may be employed to affect the efficient deliveryof nucleic acids, particularly RNAi agents, to the skin of animals. Mostdrugs are present in solution in both ionized and nonionized forms.However, usually only lipid soluble or lipophilic drugs readily crosscell membranes. It has been discovered that even non-lipophilic drugscan cross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers canbe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Such compounds are well known in the art.

Certain compositions may also incorporate carrier compounds in theformulation. As used herein, “carrier compound” or “carrier” can referto a nucleic acid, or analog thereof, which is inert (i.e., does notpossess biological activity per se) but is recognized as a nucleic acidby in vivo processes that reduce the bioavailability of a nucleic acidhaving biological activity by, for example, degrading the biologicallyactive nucleic acid or promoting its removal from circulation. Thecoadministration of a nucleic acid and a carrier compound, typicallywith an excess of the latter substance, can result in a substantialreduction of the amount of nucleic acid recovered in the liver, kidney,or other extracirculatory reservoirs, presumably due to competitionbetween the carrier compound and the nucleic acid for a common receptor.For example, the recovery of a partially phosphorothioate dsRNA inhepatic tissue can be reduced when it is coadministered withpolyinosinic acid, dextran sulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid.

The compositions can additionally contain other adjunct componentsconventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions cancontain additional, compatible, pharmaceutically-active materials suchas, for example, antipruritics, astringents, local anesthetics oranti-inflammatory agents, or can contain additional materials useful inphysically formulating various dosage forms of the compositions, such asdyes, flavoring agents, preservatives, antioxidants, opacifiers,thickening agents, and stabilizers. However, such materials, when added,should not unduly interfere with the biological activities of thecomponents of the compositions. The formulations can be sterilized and,if desired, mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings, and/oraromatic substances; and the like which do not deleteriously interactwith the nucleic acid(s) of the formulation.

The RNAi agent and additional therapeutic agents may be administered atthe same time and/or in the same combination, e.g., parenterally, or theadditional therapeutic agent can be administered as part of a separatecomposition or at separate times and/or by another method known in theart or described herein.

The in vivo methods may include administering to a subject a compositioncontaining an RNAi agent, where the RNAi agent includes a nucleotidesequence that is complementary to at least a part of an HNF4α-P2 isoformmRNA of the mammal to be treated. When the organism to be treated is amammal such as a human, the composition can be administered by any meansknown in the art including, but not limited to oral, intraperitoneal, orparenteral routes, including intracranial, intraventricular,intraparenchymal, intrathecal, intravenous, intramuscular, subcutaneous,transdermal, airway (aerosol), nasal, rectal, and topical (includingbuccal and sublingual) administration. The compositions may beadministered by intravenous infusion or injection. The compositions maybe administered by subcutaneous injection. Administration may be via adepot injection. The administration may be via a pump. The pump may bean external pump or a surgically implanted pump, such as asubcutaneously implanted osmotic pump or an infusion pump. An infusionpump may be used for intravenous, subcutaneous, arterial, or epiduralinfusions. The infusion pump may be a subcutaneous infusion pump. Thepump may be a surgically implanted pump that delivers the RNAi agent tothe liver.

Pharmaceutical compositions described herein include (a) one or moreiRNA compounds and (b) one or more agents which function by a non-RNAimechanism and which are useful in treating an HNF4α-P2 isoformmRNA-associated disorder. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit hightherapeutic indices are preferred.

Also described herein are methods of using an RNAi agent and/or acomposition containing an RNAi agent to reduce and/or inhibit HNF4α-P2isoform mRNA expression in a cell. The methods include contacting thecell with an RNAi agent described herein and maintaining the cell for atime sufficient to obtain degradation of the HNF4α-P2 isoform mRNA,thereby inhibiting expression HNF4α-P2 isoform mRNA in the cell.Reduction in gene expression can be assessed by any methods known in theart. For example, a reduction in the expression of HNF4α-P2 isoform mRNAmay be determined by determining the mRNA expression level HNF4α-P2isoform mRNA using methods routine to one of ordinary skill in the art,e.g., northern blotting, qRT-PCR; by determining the protein level ofHNF4α-P2 isoform mRNA using methods routine to one of ordinary skill inthe art, such as western blotting, immunological techniques. A reductionin the expression of HNF4α-P2 isoform mRNA may also be assessedindirectly by measuring a decrease in biological activity of HNF4α-P2isoform mRNA.

In the methods, the cell may be contacted in vitro or in vivo, e.g., thecell may be within a subject. A cell suitable for treatment using themethods described herein may be any cell that expresses an HNF4α-P2isoform mRNA. A cell suitable for use in the methods described hereinmay be a mammalian cell, e.g., a primate cell (such as a human cell or anon-human primate cell, e.g., a monkey cell or a chimpanzee cell), anon-primate cell (such as a cow cell, a pig cell, a camel cell, a llamacell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster,a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, alion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell(e.g., a duck cell or a goose cell), or a whale cell. The cell may be ahuman cell, e.g., a human liver cell.

HNF4α-P2 isoform mRNA expression is inhibited in the cell by at leastabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or about 100%. HNF4α-P2 isoform mRNA expression may beinhibited by at least 20%. The HNF4α-P2 isoform mRNA expression isinhibited selectively with respect to HNF4α-P1 isoform mRNA expression,meaning the expression of HNF4α-P2 isoform mRNA is inhibited to agreater extent, e.g., a statistically significantly greater extent, thanexpression of HNF4α-P1 isoform mRNA, e.g., by at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, orabout 100%, such as at least 20% greater extent.

An RNAi agent described herein may be administered as a “free RNAiagent.” A free RNAi agent is administered in the absence of apharmaceutical composition. The naked RNAi agent may be in a suitablebuffer solution. The buffer solution may comprise acetate, citrate,prolamine, carbonate, or phosphate, or any combination thereof. Thebuffer solution may be phosphate buffered saline (PBS). The pH andosmolarity of the buffer solution containing the RNAi agent can beadjusted such that it is suitable for administering to a subject.

Also provided herein are methods for the use of an RNAi agent or apharmaceutical composition thereof, e.g., for treating a subject thatwould benefit from reduction and/or inhibition of HNF4α-P2 isoform mRNAexpression, e.g., a subject having liver disease, liver damage, liverinflammation, or liver failure, such as acute liver failure, ALD, AH, orACLF, in combination with other pharmaceuticals and/or other therapeuticmethods, e.g., with known pharmaceuticals and/or known therapeuticmethods, such as, for example, those which are currently employed fortreating these disorders. For example, an RNAi agent targeting HNF4α-P2isoform mRNA may be administered to a subject having a liver disease,liver damage, liver inflammation, or liver failure, such as acute liverfailure, ALD, AH, or ACLF in combination with, e.g., antioxidant agents(such as N-Acetyl Cysteine), anti-inflammatory drugs (prednisolone,anakinra), liver-support devices (ELAD), high volume plasma exchange,human albumin, growth factors (G-CSF), or vitamins (vitamin K).

Examples

This human-based translational study combined integrated multi-OMICsfrom a large cohort of human samples along with in vitro andexperimental animal models with a goal to address this knowledge gap.

Identification of molecular drivers is hampered by the lack of suitableanimal models. By performing RNA-seq in livers from patients withdifferent phenotypes of alcohol-induced liver disease (ALD), developmentof AH is seen to be characterized by defective activity ofliver-enriched transcription factors (LETFs). AH was associated by amarked decrease in HNF4α-depending gene expression along with a markedexpression of the fetal HNF4α isoform (P2). P2 isoforms have same DNAbinding domain of P1 “adult type” HNF4α isoforms but lack the activationdomain 1 (AF1). A competitive inhibition of P2 over P1 protein variantsis shown. The inhibition of P2 isoforms with a novel siRNA molecule inprimary human hepatocytes and in hepatocyte cell lines led to animprovement on main HNF4A-depending genes, which regulate essentialliver functions such as gluconeogenesis, bile acid synthesis andsecretion, clotting factor synthesis and the urea cycle. Modulation ofHNF4α-depending gene expression by the silencing of P2 isoforms may bebeneficial to improve hepatocellular function in patients withalcohol-related Liver Failure, but also for liver failure caused bydifferent etiologies.

To uncover the mechanisms involved in progression to AH in patients withALD, a comprehensive analysis of liver RNA sequencing (RNA-seq) data wasperformed from a large series of patients with different disease stagesincluding normal liver, early alcoholic steatohepatitis (ASH), AH withliver failure and a unique set of explants from patients with AH thatunderwent urgent liver transplantation (Thursz, M. R., et al.Prednisolone or pentoxifylline for alcoholic hepatitis. N Engl J Med372, 1619-1628 (2015)). As diseased controls, patients withnon-alcoholic fatty liver disease (NAFLD), chronic hepatitis C andcompensated HCV cirrhosis were included. The principal componentanalysis (PCA) showed patient clustering according to the progressiveclinical phenotypes. Thus, while early ASH clustered along with chronichepatitis C and NASH close to normal livers, patients with AH showed amuch more deregulated transcriptome. A comparative analysis wasperformed between normal livers and different ALD phenotypes. Analyticalparameters of liver injury (i.e., AST) and hepatocellular syntheticfunction (i.e., INR, serum bilirubin and albumin) as well as clinicalscoring systems (i.e., Child-Pugh and MELD) were markedly impaired afterthe onset of AH. Unbiased clustering and Short Time Expression Miner(STEM) algorithm identified 13 profiles of gene expression across the 4selected disease stages. These profiles were grouped into 4 mainpatterns along ALD progression including compensatory transient geneexpression changes in early stages, genes progressively up ordown-regulated along disease progression or genes up or down-regulatedonly after the onset of liver failure. A detailed gene set enrichmentanalysis revealed down-regulation of genes related to basic hepatocytefunctions (i.e., metabolism of amino acids and lipids, biologicaloxidations, mitochondrial function and bile acid metabolism), while cellproliferation, extracellular matrix regulation and inflammation relatedpathways were enriched among up-regulated genes. To gain insight intothe main drivers of gene expression that could result in the developmentof hepatocellular failure in AH, the predicted activity of transcriptionfactors was analyzed using a complementary approach, by combining thesearch of transcription factor binding motifs in the promoter ofdifferentially expressed genes (DEG) and by the use of Ingenuity PathwayAnalysis (IPA) software to uncover predicted upstream transcriptionfactor activity (see Material and Methods section). Early compensatedstate of ALD was characterized by an increased predicted activity of thehepatoprotective transcription factor PPAR-γ. In contrast, developmentof AH was associated with a profound decrease in the activity of LETFs,especially HNF4α (FIG. 1A). The results obtained in human livers wereassessed in several animal models of early and advanced ALD. While amodel of early experimental ALD (HFD plus intragastric EtOH for 3 weeks)showed a marked activation of PPAR-γ, a model of severe ALD (CCL4 plusintragastric EtOH for 9 weeks) was characterized by decreased predictedactivation of FOXA-1, but not HNF4α. Preserved HNF4α function couldexplain the lack of liver failure (i.e., jaundice and coagulopathy) inmice models of severe ALD.

The correlation between parameters indicative of liver syntheticfunction and HNF4α activity was studied. As shown in FIG. 2A,development of liver failure in the setting of AH, as indicated byelevated serum bilirubin levels and INR and decreased albumin synthesis,was strongly associated with a negative HNF4α Z-Score on IPA analysis.HNF4α is known to have a fetal isoform driven by a ˜45 kb upstreamalternative promoter (P2-HNF4α). During embryonic development, the P2promoter is used and alternative splicing of the first exon is produced,originating the fetal isoforms α7-12. These variants lack the AF-1domain in the N-terminal of the protein resulting in lesstransactivation activity, affecting its interaction with co-regulators(FIG. 2B). The relevance of P2 derived isoforms in adult human liverdisease is not well-known. The expression of N-terminal isoforms wasstudied in normal and AH human livers. HNF4α-P1 mRNA remained unchangedin AH, while there was a dramatic up-regulation in the expression of thefetal HNF4α-P2 isoform in livers from patients with AH (FIG. 2C). It wasfound that the expression of the lncRNAN HNF4A-AS1, which shares the P1promoter region with HNF4A, was downregulated in patients with AH (FIG.2C). Up-regulation of HNF4α-P2 was not seen in early forms of ALD or inother types of liver diseases such as NASH and chronic hepatitis C. Inorder to further explore the regulation of the HNF4α locus, we used aspecific computational tool (Multivariate Analysis of TranscriptSplicing—MATS—) (Shen, S., et al. MATS: a Bayesian framework forflexible detection of differential alternative splicing from RNA-Seqdata. Nucleic Acids Res 40, e61 (2012)) to assess differences in HNF4αsplicing between normal and AH livers. AH livers showed increasedexpression of exon 1D, 4,5, 6,9 and 10. Correlation analysis of exonexpression suggest a profound deregulation of HNF4A gene splicing in AHnot only by the increase of P2 isoforms, but also by the decrease ofphysiological exon exclusion in C-terminal exons. At the protein level,the HNF4α-P1 signal was detected in the nuclei of both normal and AHhepatocytes. Conversely, the HNF4α-P2 isoform, barely detected in thenucleus of normal livers, was markedly up-regulated in AH hepatocytes(FIGS. 2D and 2E). Other important LETFs inhibited in AH such as HNF1αand FOXA-1 showed decreased nuclear expression and increased cytoplasmiclocalization.

Whether P2 expression in hepatocytes contributes to the loss of maturehepatocyte biological functions during AH including bile acidhomeostasis was determined, as well as metabolic and syntheticfunctions. Because primary hepatocytes undergo de-differentiation inculture, the expression of HNF4α isoforms during this process wasexamined. Gene expression of the P1 isoform did not change during thefirst 48 h, while the P2 isoform was 30-fold up-regulated at early timepoints (FIG. 2F). Phosphoenolpyruvate carboxy-kinase 1 (PCK1), therate-limiting enzyme in gluconeogenesis, and albumin (ALB) mRNA levelswere down-regulated at 24 and 48 h (FIGS. 2G and 2H). Importantly,HNF4α-P2 mRNA levels correlated negatively with genes related tohepatocyte function (such as PCK1, ALB and CYP2E1) and positively withgenes related to hepatocyte de-differentiation (such as Vimentin, Epcam,IL8 and KRT7) (FIG. 2I). In AH patients, increased expression ofprogenitor cell markers and markers of epithelial-to-mesenchymaltransition (EMT) were found, suggesting a de-differentiation ofhepatocytes, furtherly suggested by correlation analysis with tissue andcell type published gene sets. Gain and loss-of function studies wereperformed to elucidate the role of P2 in hepatocyte biologicalfunctions. Overexpression of HNF4α-P2 results in decreased expression ofthe HNF4α target gene PCK1 (FIGS. 2J and 2K). In contrast, abrogation ofHNF4α-P2 resulted in increased HNF4α-P1 gene and protein expression(FIGS. 2I-2N), without affecting the levels of HNF4A-AS1 (FIG. 2O). Theexpression of HNF4α target genes involved in hepatocyte metabolic,secretory and synthetic functions such as PCK1, coagulation factor VII(F7), ALB, CYP7A1 and biliary salt export pump (BSEP) was increased(FIGS. 2P and 2Q). Moreover, this maneuver restored bile acid synthesisand secretion (FIG. 2R) and stimulated glucose production (FIG. 2S) inhuman primary hepatocytes. Overall, these results suggest that P2overexpression negatively regulates HNF4α-dependent gene expression andseveral biological properties of mature hepatocytes that are commonlylost in AH.

The potential mechanisms involved in HNF4α P1-P2 imbalance during thedevelopment of liver failure in AH was explored. Unbiased analysis oftranscriptomic changes in patients progressing to AH uncovered mainupstream regulators. When comparing the expression of top enrichedupstream regulators through different phenotypes, TGβ1 was found to bethe most relevant factor, followed by EGF. Expression of TGβ1-relatedgenes such as TGβ1 receptors 1 and 2 and osteopontin (SPP1), as well asthe EGFR ligand Amphiregulin (AREG), were markedly increased in AHlivers. TNFα transcript levels were not specifically elevated in AH whencompared with other liver disease. It was then hypothesized that TGβ1and AREG regulate HNF4α P1-P2 relative expression in hepatocytes. TGβ1and AREG synergistically decreased HNF4α-P1 expression and increasedHNF4α-P2 through activation of TGFβ1RI/TAK1 and the EGFR/MEK/ERK axisrespectively. The effect of TGβ1 and AREG on HNF4α levels involvedproteasome-related degradation. TGβ1 reduced HNF4α-P1 target genes andthe blockage of TGFβ1RI restored the levels of rate-limiting enzymes inmature hepatocytes such as PCK1 and ornithine-transcarbamylase (OTC). Itwas then explored if the detrimental effect of TGβ1 on hepatocytefunction is mediated by HNF4α-P2 increase. Hepatocytes transfection withsiRNA targeting P2 isoforms abolished TGβ1-mediated suppression ofHNF4α-P1. The inhibition of HNF4α-P1 dependent genes, in particularCYP7A1 and BSEP, was also significantly prevented by P2 silencing (FIGS.3A-3E). These results suggest that the re-expression of HNF4α fetalisoforms in AH could participate in TGβ1-induced loss of hepatocellularfunction, pointing to these isoforms as potential therapeutic targets.

Next, potential mechanisms were identified that maintain the normalHNF4α P1/P2 ratio during the compensated stages of ALD. Transcriptomicfootprint analysis revealed a marked predicted activation of PPAR-γ inearly phases of ALD. Whether PPAR-γ antagonizes TGβ1-mediated HNF4αdysregulation was explored. The PPAR-γ agonists rosiglitazone andpioglitazone decreased the abundance of P2 isoforms and increased P1isoforms (FIG. 3F). The effect on P2 protein expression, but not P1, wasregulated post-transcriptionally. The effect of rosiglitazone onHNF4α-P1 mRNA levels was dose dependent. TGβ1-mediated ALBdown-regulation was restored by PPAR-γ activation. Overall, theseresults suggest that, in hepatocytes, PPAR-γ counteracts TGβ1-mediatedHNF4α dysregulation.

Finally, whether genetic or epigenetic factors are involved in thedefective LETFs function in AH was explored. To address this question,GWAS data was first analyzed from a large cohort of AH patients andpatients with ethanol abuse but never decompensated. None of the singlenucleotide polymorphisms (SNP) detected in LETFs including HNF4α, eithergenotyped or imputed, were significantly associated with AH development.Because exposure to either TGβ1 or alcohol have been involved in DNAmethylation and chromatin remodeling, it was hypothesized that thedisruption of the expression and activity of the transcriptional masterregulators (i.e., LETFs) in patients with AH could be part of a globalepigenetic remodeling. A disease-specific increase was found in theexpression of genes encoding DNA methyl transferases DNMT1 and DNMT3A,histone deacetylases HDAC7 and SMARC4 and histone acetyl transferasesKAT6A and KAT6B. The methylation status of nearly 800,000 loci in normallivers and livers from AH patients was analyzed and found around 3,000differentially methylated (DM) CpG-containing loci. Motif enrichmentanalysis of DM regions revealed the presence of HNF4α and PPAR-γ motifsin hypermethylated regions while hypomethylated regions were enriched inmotifs of inflammatory transcriptional regulators, such as STAT4 and AP1complex (c-FOS, JUN). The analysis of DM-CpG nearest genes withIngenuity Pathway Analysis showed that among hypermethylated regionsHNF4α footprint was the most enriched transcriptional regulator. Theseresults mirrored data from RNA-seq analysis, showing a parallel betweenhypermethylation and down-regulation of regions controlled by LETFs(e.g., HNF4α, HNF1α, CEBPα, SREBPs, CEBPβ), and other hepatoprotectivefactors such as PPAR-γ (FIG. 4i-k ). The analysis of soluble upstreamregulators revealed TNFα and TGβ1 involvement in the expression of genescontaining hypomethylated CpG (FIG. 4l ). Lastly, data from H3K27Acchromatin immunoprecipitation coupled to DNA sequencing (ChIP-seq) ofnormal livers and livers from patients with AH was analyzed. Thischromatin mark is known to be enriched in active regulatory regions.Promoter regions of HNF4α targets such as PCK1, CYP3A4 and F7 were poorin H3K27Ac, whereas other gene promoter targets of RELA, like BCL2L1 andICAM1 were rich in this mark. When focusing on HNF4A genomic locus,enhanced H3K27Ac mark was found in the P2 promoter, in accordance withour RNA expression results. Finally, whether the defectiveLETFs-depending gene expression in livers with AH results in an abnormalplasma footprint of the corresponding proteins was explored. Plasma wascollected from controls and patients with AH and performed massspectrometry. Among the 288 plasma proteins detected in plasma of bothcontrols and AH patients, 60 corresponded to liver-secreted proteinswhose gene expression was altered in AH livers. Importantly, 21 of theseproteins belong to the footprint of LETFs altered in AH and correlatedwith hepatic gene expression. These peripheral footprints could beuseful for prognosis, patient stratification or personalized treatmentallocation in future clinical trials.

In conclusion, this human-based translational study found that thedevelopment of hepatocellular failure in patients with AH ischaracterized by a dramatic decrease in HNF4α-depending gene expression.The mechanisms likely involve TGβ1 that induces the use of HNF4α P2promoter in hepatocytes, an effect attenuated by PPAR-γ agonists. Genepolymorphisms in LETFs including HNF4α do not predispose to thedevelopment of AH, while AH livers are characterized by profound changesin DNA methylation state and chromatin remodeling in HNF4α-dependentgenes. The results of this study suggest that targeting TGβ1 andepigenetic drivers that modulate HNF4α-dependent gene expression couldbe beneficial in patients with AH.

Materials and Methods Human RNAseq Studies

Human liver samples were obtained from the Human Biorepository Core fromthe NIHfunded international InTeam consortium (7U01AA021908-05).Patients with early alcoholic steatohepatitis (ASH) were obtained fromCliniques Universitaires Saint-Luc (Brussels, Belgium). All patientsincluded gave written informed consent and the research protocols wereapproved by the local Ethics Committees. A total of 76 patients wereincluded. Patients were selected according to different clinicallyrelevant stage groups: 1) patients with early ASH, who were non-obesewith high alcohol intake, and presented mild elevation of transaminasesand histologic criteria of steatohepatitis (ASH, N=12); 2) patients withhistologically confirmed alcoholic hepatitis (AH) who were biopsiedbefore any treatment (AH, N=18) and 3) explants from patients with AHwho underwent early transplantion following a well-defined protocol¹(exAH, N=10). These groups were compared with fragments of non-diseasedhuman livers (N=10), patients with nonalcoholic fatty liver disease(NAFLD) according to Keiner's Crieria² and without alcohol abuse (N=9)and from patients with non-cirrhotic HCV infection (N=9) and compensatedHCV-related cirrhosis (N=9). Patients with malignancies were excludedfrom the study. A selection of liver samples from patients with AH (N=6)and fragments of normal human livers (N=5), were used for Methylome andChIP seq analysis.

Patients for IHCs Analyses

Patients with normal liver and AH were obtained at the Division ofGastroenterology and Hepatology, Medical University of Graz, Austria.All patients had clinically and histologically confirmed AH (N=10) anddid not have any concomitant causes of chronic liver disease (N=10)⁴.The study was approved by the Ethics Committee of the Medical Universityof Graz and performed in accordance with the Declaration of Helsinki.

RNA Extraction, Sequencing and Bioinformatic Analysis

Total RNA from flash-frozen liver tissue was extracted byphenol/chloroform separation (TRIzol, Thermox). RNA purity and qualitywere assessed by automated electrophoresis (Bioanalyzer, Agilent) andwas sequenced using Illumina HiSeq2000 platform. Libraries were builtusing TruSeq Stranded Total RNA Ribo-Zero GOLD (Illumina). Sequencingwas paired end (2×100 bp) and multiplexed. Ninety-four paired-endsequenced samples obtained an average of 36.9 million total reads with32.5 million (88%) mapped to GRCh37/hg19 human reference. Short readalignment was performed using STAR alignment algorithm with defaultparameters⁵. To quantify expression from transcriptome mappings weemployed RSEM⁶. Principal component analysis (PCA) was done using made4library⁷. Analysis of differential expression was performed using theLimma package⁸. Cyclic loess normalization was applied, followed by logtransformation of the counts per million and mean-variance adjustmentusing the voom function. The Jonckheere-Terpstratest and Kendallcorrelation was used to check ordered differences gene among progressivedisease stages. To agglomerate gene patterns along disease stages, ShortTime-course Expression Miner (STEM) algorithm was used through on-lineplatform⁹. To uncover biological functions related to gene expressionchanges, Gene Ontology (GO) enrichment through gene set overlappingcomputation was done by means of Molecular Signatures Database v3.0,using the Canonical Pathways (CP) collection, which includes 1,329 genesets¹⁰. To identify in an unbiased way the transcription factorspredicted to be directly involved on transcriptomic changes we apply twomethods: 1) Transcription factor motif searching in gene promoters andproximal 5′ regulatory regions (−/+2,000 bp from TSS) by means of 2)Opossum on-line tool^(11 and 2)) Functional prediction of differentiallyexpressed genes (DEG) by the use of Ingenuity Pathway Analysis (IPA,Qiagen), selecting among predicted upstream regulators, those involvedin transcriptional regulation (categories: “transcriptional regulator”,“ligand-dependent nuclear receptor”). Only those hits found in bothanalyses were considered. The statistic approach used to calculate thepredicted activation state (IPA) was Z Score (ZS) and is used to inferlikely activation states of upstream regulators based on comparison witha model that assigns random regulation directions. An overlap p-value todetermine statistically significant overlap between the transcriptionfactor target gene dataset and the DEG of each comparison was alsocalculated, using Fisher's Exact Test. For this study, all the selectedtranscription factors (FIGS. 1b and 3f ) shown an overlap p value <0.01(data not shown). Opossum calculates two complementary scoring methodsto measure the over-representation of transcription factor bindingsites: (1) Z-scores measures the change in the relative number of TFBSmotifs in the DEG gene set compared with the background set, and (2)Fisher scores based on a one-tailed Fisher exact probability assessingthe number of genes with the TFBS motifs in the foreground set vs. thebackground set. JASPAR database was used as the source of DNA bindingprofiles.

HNF4a Gene Splicing Analysis

RNA-seq reads were trimmed to a uniform length of 75 bp using theFastxToolkit (http://hannonlab.cshl.edu/fastx_toolkit/). After readtrimming, alignment of RNA-seq reads was performed with the STAR aligner(v2.5.2a) against the hg19 human genome. Resulting bam files wereindexed with samtools for rMATS as described previously¹². Differentialexpression of splice isoforms was completed using STARalignment-StringTie-BallGown pipeline as described elsewhere¹³. Toidentify exon-specific expression, an alternate pipeline was used.First, reads were put through adapter trimming using TrimGalore(https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Afterthe trimming, reads were aligned with the STAR aligner (v2.5.2a) againstthe hg19 genome. The resulting bam files were then put through theDEXSeq R Bioconductor package (v1.26.0 for DEXSeq and 3.3.1 for R)pipeline. To obtain raw read counts for each exon, we used a standardDEXSeq script for exon counting (dexseq_count.py), with minormodifications. The exons were categorized in the GenCode v19 release.After exon counting, individual R scripts were used to obtain theexon-specific expression profiles. All custom scripts are available uponrequest.

Genomic DNA Methylome Analysis

Genomic DNA (gDNA) was extracted from flash-frozen liver tissue withPureLink Genomic DNA Mini Kit (Thermo) and quantified using Nanodrop(Thermo). 1 μg of isolated gDNA was bisulfite converted, denatured,fragmented and hybridized to Infinium Methylation Bead Chip, followingthe manufacturer protocol (Infinium MethylationEPIC kit, Illumina).BeadChips were imaged using an Illumina Scan System and intensity wasdetermined by iScan Control Software (Illumina). Sample intensities werenormalized using functional normalization from the minfi package(v1.24.0)¹⁴. Probes failing a detection p-value threshold (0.01) in atleast 50% of samples were removed, as were probes identified ascontaining a SNP with a MAF >0.05. Differentially methylated probes wereidentified by applying limma (v3.34.3)⁸ contrasts to M values (absolutechange in beta value >0.1, FDR-corrected Pvalue <0.05). Differentiallymethylated regions were identified using DMRcate (v1.14.0)¹⁵ setting athreshold of absolute change in beta value in >0.1 and of Stouffer'svalue in <0.05.

Chromatin Immunoprecipitation-Deep Sequencing (ChIP-Seq)

ChIP-seq was performed in Mayo Epigenomics Development Laboratory aspreviously described¹⁶. ChIP-seq with the liver tissue from 5 controlsand 7 severe AH explants (provided by University of Lille, France) weredone for four histone modifications, including histone H3 acetylation(H3K27ac with anti-H3K27ac antibody (CST, #8173)). For thenext-generation sequencing, ChIP-seq libraries were prepared from 10 ngof ChIP and input DNAs with the Ovation Ultralow DR Multiplex system(NuGEN). The ChIP-seq libraries were sequenced to 51 base pairs fromboth ends using the Illumina HiSeq 2000 in the Mayo Clinic MedicalGenomics Core. Data were analyzed by the HiChIP pipeline¹⁷. Briefly,reads were aligned to the hg19 genome assembly using BWA and visualizedusing the Integrative Genomics Viewer (IGV). Mapped reads werepost-processed to remove duplicates and pairs of reads mapping tomultiple locations. The MACS2 and Sicer algorithm was used forpeak-calling in relation to the input DNA. IGV was then used tovisualize H3K27ac peak changes on individual genes in this study.

Human Primary Hepatocytes and Cell Lines

Primary human hepatocytes were purchased from Lonza. They were thawed inthawing medium (MCHT, Lonza), plated in plating medium (MP, Lonza), andcultured in maintenance medium (MM, Lonza). PHH were seeded oncollagen-coated 12- or 6-well plates (Corning), allowed to attach for 4hours, and then overlaid with Matrigel (0.3 mg/mL; Corning). Insilencing experiments, transfection was done 6 h before Matrigel overlayand cells were kept in reduced serum media (OptiMEM, Gibco) during thattime. Cells and/or supernatant were collected at the indicated timepoints. HepG2 cells and Hep3B cells were purchased from ATCC and weremycoplasma-free. They were expanded in Dulbecco's Minimum EssentialMedia (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (FBS,Gibco), 1 unit/mL Penicillin (Gibco), and lug/mL Streptomycin (Gibco).When indicated, cells were serum-starved (1% FBS DMEM) 2 h prior drugincubation. In silencing experiments, transfection was done 24 h or 48 hbefore treatment and cells were kept in OptiMEM for 6 h aftertransfection and then in 1% FBS DMEM until harvesting.

RNA Extraction and Real Time Polymerase Chain Reaction (RT-PCR)

RNA from human biopsies, for RT-PCR experiments was extracted withQiagen AllPrep DNA/RNA/Protein kit (Qiagen) following manufacturer'sinstructions. For experiments with cell lines and primary hepatocytes,RNA was extracted by phenol/chloroform method (TRIzol, Invitrogen).Concentration and purity was assessed by spectrophotometry (Nanodrop,Thermo). 1 μg of total RNA was used for reverse transcription reactionusing Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase(Thermo) following manufacturer protocol. RT PCR of 50 ng of cDNA wasperformed in a 96 well plate, using a CFX96 Real Time PCR detectionsystem (BIO-RAD) and fluorescent double-stranded DNA-binding dye(SsoAdvanced Universal Sybr Green Supermix, BIO-RAD). The comparative CTmethod (2-ΔΔCt) was used to determine fold changes in mRNA expressioncompared a control group after normalization to an endogenous referencegene (Human Histone3B3).

Protein Extraction and Western Blot

Liver tissue fragments and cell pellets were lysed in RIPA buffer (150mM NaCl, 50 mM Tris pH 7.5, 0.1% SDS, 1% Triton X-100) with the additionof 40 mM DTT, protease inhibitor cocktail (Complete, Roche) andphosphatase inhibitors (1 mM Na₃VO₄, 2 mM NaF and 2 mMb-glycerophosphate) just before protein extraction. For liver extracts,ratio 1:20 (mg:V) was used, and tissue was pestle and sonicated (5cycles of 20 sec with a probe sonicator at 20% Amplitude). In indicatedcases, nuclear/cytoplasm fractionation was made by using the NE-PER kit(Thermo), following the manufacturer protocol. For western blot, 20-40ug of protein extract was denatured with Laemli buffer (AlfaAesar),boiled (95° C. for 3 min), loaded in SDS-PAGE system (BIO-RAD), rununtil complete separation, transferred to a nitrocellulose membrane (0.2um pore diameter, BIO-RAD). Membranes were blocked for 1 h at roomtemperature with 5% non-fat milk in 0.1% Tween20-Tris Buffered Saline(T-TBS). After overnight incubation with primary antibodies, membraneswere washed three times with T-TBS and incubated with Near-InfraredFlorescent secondary antibodies (IRDye 680CW Goat anti-Rabbit and/orIRDye 800CW Goat anti-Mouse, LiCOR) for 1 h at room temperature andwashed twice with T-TBS and finally rinsed with TBS. Membranes wereimaged using an Odissey CLx Imager (LiCOR).

Silencing and Overexpression of HNF4a Isoforms

For silencing of HNF4A-P2 isoforms, a specific custom siRNA and itsScramble (Scr) were generated by using BLOCK-iT RNAi Designer onlinetool (Thermo-Fisher). The sense-strand sequence of siRNA anti-HNF4A-P2was GCTCCAGTGGAGAGTTCTTdTdT and that of Scr wasGCTGAGTAGAGTGTCCCTT-dTdT. The effective working concentration of siRNAwas 20 pM in primary hepatocytes and 10 pM in HepG2/Hep3B cells.Transfection of siRNAs was performed by the use of Lipofectamine-RNAiMAX(Invitrogen) following the manufacturer recommendations. This protocolshowed 70-85% of silencing efficiency (mRNA and Protein level) at 24and/or 48 h. For overexpression of HNF4a-P1 dependent isoforms, ORF ofHNF4a2 was cloned in a pcDNA6 (Invitrogen) under the CMV promoter.Plasmid containing HNF4a8 ORF under CMV promoter, was gently providedfrom Dr. Bell (Pittsburgh Liver Research Center). Plasmids weretransfected at the indicated doses in HepG2 cells using Lipofectamine3,000 (Invitrogen) following standard manufacturer protocol.

Cell Culture Treatments

TGFb1 (5 ng/mL, R&D Systems) or amphiregulin (AREG, 50 nM, SigmaAldrich) were added immediately before Matrigel overlay and mRNA orprotein were collected at the indicated time points. For was used. Forproteasome inhibition, MG132 (10 μM, Calbiochem-EMD Millipore) was added45 min prior to cell harvesting. Treatments with TGF-β RI KinaseInhibitor VI (5 nM, SB431542, Calbiochem-EMD Millipore), TAKI Inhibitor(0.5 or 1 μM, NG25 trihydrochloride, Axon), EGFR inhibitor (3 μM,PD153035, Calbiochem-EMD Millipore), MEK Inhibitor (10 μM, U0126,Promega), PPARg agonists, Rosigiltazone (10 μM, Sigma) and Pioglitazone(10 μM, Sigma), and Dexamethasone (0.25 μM, Sigma), was performed afterof 2 h starvation (1% FBS DMEM) and 45 min before TGFb1 treatment.

Biliary Acid Quantification

Cryopreserved human primary hepatocytes (Lonza) were plated overnight oncollagencoated 96-well plates at 2×104 cells per well in MM (Lonza) andcollected after 24 and 48 h of siRNA transfection. Total bile acids weremeasured following the protocol supplied in the Total Bile Acid AssayKit available from Cell Biolabs (San Diego, Ca). Absorbance data wascollected using the SpectraMax M2 (Molecular Devices, Sunnyvale, Calif.,USA) microtiter plate reader. The total bile acids were calculated byextrapolating test values to a calibration curve as described in theassay kit.

Glucose Production Assay

Cryopreserved human primary hepatocytes (Lonza) were plated overnight oncollagencoated 12-well plates at 1×105 cells per well in MM (Lonza).Twenty-four hours after plating, cells were serum-starved in DMEM basemedium (Sigma) supplemented with 1 g/L glucose (Sigma), 3.7 g/L sodiumbicarbonate (Sigma), and 4 mM L-glutamine (Corning) overnight, followedby 24 hours incubation in 0.3 ml glucose-production medium: DMEM basewith 2 mM glutamine, 3.7 g/L sodium bicarbonate, 15 mM HEPES(ThermoFisher), 20 mM lactate (Sigma), 2 mM pyruvate (Fisher) and 0.1 mMpCPT-cAMP (Sigma). After 24 hours, 50 pL of medium was removed forglucose detection with Invitrogen Glucose Colorimetric Detection kit(#EIAGLUC), according to manufacturer's protocol, and read on a platereader (Multiskan G O, Thermo-Scientific).

Model of Alcoholic Liver Disease

Animals: Male mice (C57BL/6J, 20-25 g, 12 weeks of age) were obtainedfrom the Jackson Laboratory (Bar Harbor, Me.) and housed in atemperature-controlled environment with a 12-h light-dark cycle and weregiven free access to regular laboratory chow diet and water. All studieswere approved by the Institutional Animal Care and Use Committee atUNC-Chapel Hill.

Diets and Treatment: CCl4 (>99.5% pure) and olive oil vehicle were fromSigma (St. Louis, Mo.), ethyl alcohol (EtOH) (190 proof, Koptec) wasfrom VWR (Radnor, Pa.). Procedures for CCl4-induced liver fibrosis wereas detailed elsewhere¹⁸. Mice were intraperitoneally injected (15 ml/kg)with CCl4 (0.2 ml/kg) or olive oil vehicle-alone 2×week for 6 weeks.After 6 weeks of CCl4 treatment, animals underwent surgical intragastricintubation¹⁹. Following surgery, mice were housed in individualmetabolic cages and allowed one week to recover with ad libitum accessto food and water. Animals had free access to water and non-nutritiouscellulose pellets throughout the remaining study. Alcohol groupsreceived high-fat diet containing ethyl alcohol as detailed elsewhere¹⁹.Alcohol was delivered continuously through the intragastric cannulainitially at 16 g/kg/day and was gradually increased to 25 g/kg/day. Allanimals were given humane care in compliance with the NationalInstitutes of Health guidelines and alcohol intoxication was assessed toevaluate the development of tolerance. At the end of the study, micewere anesthetized with pentobarbital (50 mg/kg, i.p.) and sacrificed viaexsanguination through the vena cava, which was the site of bloodcollection. Tissues were excised and snap-frozen in liquid nitrogen.

Immunohistochemistry

Dewaxed 3 μm thick sections were stained with hematoxylin and eosin(H&E) or chromatrope aniline blue (CAB) connective tissue stainaccording to standard protocols. All slides were reviewed by a singlepathologist (CL). For immunohistochemistry paraffin sections weredewaxed and rehydrated. After immunohistochemical staining sections werecounterstained with hematoxylin (Labonord, Templemars, France) andmounted with Aquatex (Merck, Darmstadt, Germany). Immunohistochemicalsignals were evaluated semi-quantitatively by the application ofnumerical scores, based on the intensity of the signal. For HNF4a, HNF1aand FOXA1, where the signal in AH patients was also cytoplasmic, scoringwas made separately for cytoplasmatic and nuclear signals.

Single Nucleotide Polymorphism (SNP) Analysis

Patients with AH (n=878) were recruited through the steroids orpentoxifylline for alcoholic hepatitis (STOPAH) trial²⁰. Inclusion wasbased upon a clinical diagnosis of alcoholic hepatitis, modifiedMaddrey's discriminant (mDF) ≥32, current excess alcohol consumption,recent onset of jaundice and exclusion of other causes of decompensatedliver disease²¹. Controls with a background of alcohol dependence butwith no evidence of liver injury were recruited via the UniversityCollege London Consortium (N=318). All were of English, Scottish, Welshor Irish descent with a maximum of one grandparent of white EuropeanCaucasian origin. None of the individuals was related. The alcoholiccirrhosis study data were obtained from a genome-wide association studyof alcohol-related cirrhosis with data from a total of 2,178individuals²². Publicly available study summary data were downloadedfrom gengastro.med.tudresden. de/suppl/alc_cirrhosis/. There was knownoverlap between the control populations of the alcoholic hepatitis studyand United Kingdom cohort of the alcoholic cirrhosis study. Samples weregenotyped using the HumanCoreExome beadchip (Illumina) at the WellcomeTrust Sanger Institute (Cambridge, UK). Quality control and analysis ofdata were performed in PLINK v1.90. Individual data were qualitycontrolled such that those with genotyping rate <98%, sampleheterozygosity >3 standard deviations from the population mean,relatedness determined by pi-hat >0.185 or phenotypic and genotypic sexmismatch were excluded. Markers with genotyping rate <98% or with aprobability of deviation from Hardy-Weinberg equilibrium <10⁶ were alsoexcluded. Population principal components were calculated using alinkage-disequilibrium pruned data set of common variants in PLINKv1.90, associations between principal components and case-control statuswere tested using R. The principal components associated withcase-control status were specified as covariates in analyses. Keytranscription factors and related genes were identified through theprimary analysis of RNA-seq data. Genomic coordinates for the codingregions of these genes were obtained from ensembl Biomart. Singlenucleotide polymorphisms (SNPs) falling within these genetic loci wereextracted from the alcoholic hepatitis study data. Analyses were limitedto SNPs with a minor allele frequency >1%. Study significance was setthrough application of a Bonferroni correction for the number of testsperformed for a=0.05. For significantly associated SNPs predictedeffects on protein structure were predicted using SIFT²³ and Polyphen²⁴,expression quantitative train locus (eQTL) tests were conducted usingGTeX²⁵. The SNPs most significantly associated with disease risk at eachlocus were further examined in the alcohol-related cirrhosis dataset.Gene- and pathway-based association tests were performed using studysummary statistics in MAGMA v1.06²⁶. Study-specific significancethreshold was set using a=0.05 corrected for the number of genesevaluated using the Bonferroni method. Pathway-based association testingwas achieved by defining a biological pathway incorporating the genetarget of interest.

Mass Spectrometry of Plasma Samples (LC-MS/MS)

Plasma samples from Control subjects (N=10, 10 μL each) and plasma frompatients with AH (N=15, 10 μL each) were pooled and proteinconcentration of each group was determined by Qubit fluorometry. 10μL ofprotein from each pooled sample was depleted in duplicate on a Pierce™Top 12 Abundant Protein Depletion Spin Column (Thermo Scientific)according to manufacturer's protocol. Depleted samples were bufferexchanged into water on a centrifugal concentrator (Spin X, Corning)using a 5 kD molecular weight cut off and quantified by Qubitfluorometry (Life Technologies). 50 μg of each sample was reduced withdithiothreitol, alkylated with iodoacetamide and digested overnight withtrypsin (Promega). The digestion was terminated with formic acid. Eachdigested sample was processed by solid phase extraction using an EmporeC18 (3M) plate under vacuum (5 in Hg). Briefly, columns were activatedwith 400 μL 95% acetonitrile/0.1% TFA×2, and then equilibrated with 400μL 0.1% TFA×4. Acidified samples were samples were loaded and columnswere washed with 400 μL 0.1% TFA×2. Peptides were eluted with 200 μL 70%acetonitrile/0.1% TFA×2 and then lyophilized for further processing. 2μg of each sample was analyzed by nano LC-MS/MS with a NanoAcquity HPLCsystem (Waters) interfaced to a Q Exactive (Thermo-Fisher). Peptideswere loaded on a trapping column and eluted over a 75 μm analyticalcolumn at 350 nL/min using a 3 hr reverse phase gradient. Columns werepacked with Luna C18 resin (Phenomenex). The mass spectrometer wasoperated in data-dependent mode, with the Orbitrap operating at 60,000FWHM and 17,500 FWHM for MS and MS/MS respectively. The fifteen mostabundant ions were selected for MS/MS. Data were searched using a localcopy of Mascot with the following parameters: Enzyme: Trypsin/P;Database: SwissProt Human. Fixed modification: Carbamidomethyl (C);Variable modifications: Oxidation (M), Acetyl (N-term), Pyro-Glu (N-termQ), Deamidation (N/Q); Mass values: Monoisotopic; Peptide MassTolerance: 10 ppm; Fragment Mass Tolerance: 0.02 Da; Max MissedCleavages: 2. Mascot DAT files were parsed into Scaffold (ProteomeSoftware) for validation, filtering and to create a non-redundant listper sample. Data were filtered using at 1% protein and peptide FDR andrequiring at least two unique peptides per protein. Normalized SpectralAbundance Factor (NSAF) values were used to obtain the fold changebetween Normal and AH groups. For unbiased searching of secreted proteincoding genes from RNA-seq data, Retrieve/ID mapping online tool ofUniProt was used (filters “signal peptide” and “NOT transmembranedomain”)²⁷.

REFERENCES (MATERIAL & METHODS)

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The following numbered clauses describe various aspects or embodimentsof the present invention:

Clause 1: A method of treating a patient having liver damage or liverfailure, comprising knocking down or inhibiting expression of ahepatocyte nuclear factor 4 alpha mRNA transcribed from its P2 promoter(HNF4α-P2 isoform mRNA) in the patient, or reducing activity of theprotein encoded by the HNF4α-P2 isoform mRNA, thereby treating the liverdamage or liver failure in the patient.

Clause 2: The method of clause 1, wherein the liver damage or liverfailure is acute liver failure.

Clause 3: The method of clause 1, wherein the liver damage or liverfailure is associated with alcohol-related liver disease (ALD),alcoholic hepatitis (AH), or Acute-on-Chronic Liver Failure (ACLF).

Clause 4: The method of any one of clauses 1-3, wherein the treatmentnormalizes or decreases blood bilirubin levels, normalizes or decreasesprothrombin time, and/or normalizes or increases serum albumin.

Clause 5: The method of any one of clauses 1-4, wherein an RNAi agentfor knocking down or inhibiting expression of an HNF4α-P2 isoform mRNAis administered to the patient in an amount effective to treat the liverdamage or liver failure in a patient.

Clause 6: A method of knocking down expression of an HNF4α-P2 isoformmRNA in a cell, comprising contacting the cell with an RNAi agent forselectively knocking down expression of an HNF4α-P2 isoform mRNA, in anamount effective to reduce production of the protein product of theHNF4α-P2 isoform mRNA in a cell.

Clause 7: The method of clause 6, wherein the cell is a liver cell.

Clause 8: The method of clause 6 or 7, wherein the cell is a human cell.

Clause 9: The method of any one of clauses 6-8, wherein the cell is invitro.

Clause 10: The method of any one of clauses 6-8, wherein the cell is invivo.

Clause 11: The method of any one of clauses 5-10, wherein the RNAi agenttargets a contiguous sequence of 15 or more bases within bases 1-63, orwithin bases 1-53 of SEQ ID NO: 1.

Clause 12: The method of clause 11, wherein the RNAi agent targets acontiguous sequence of 15 or more bases within the sequence:GCTCCAGTGGAGAGTTCTTACGACACG (SEQ ID NO: 1, bases 32-58).

Clause 13: The method of clause 12, wherein the RNAi agent targets thesequence: GCTCCAGTGGAGAGTTCTT (SEQ ID NO: 1, bases 32-50),CTCCAGTGGAGAGTTCTTA (SEQ ID NO: 1, bases 33-51), TCCAGTGGAGAGTTCTTAC(SEQ ID NO: 1, bases 34-52), or GGAGAGTTCTTACGACAC (SEQ ID NO: 1, bases40-57).

Clause 14: The method of any one of clauses 5-11, wherein the sensestrand of the RNAi agent has the sequence GCTCCAGTGGAGAGTTCTTdTdT (SEQID NO: 30), CTCCAGTGGAGAGTTCTTAdTdT (SEQ ID NO: 32),TCCAGTGGAGAGTTCTTACdTdT (SEQ ID NO: 34), or GGAGAGTTCTTACGACACdTdT (SEQID NO: 36).

Clause 15: The method of any one of clauses 5-11, wherein the RNAi agentis chosen from GCTCCAGTGGAGAGTTCTTdTdT (SEQ ID NO: 30),CGTGTCGTAAGAACTCTCCdTdT (SEQ ID NO: 31), gscstccaGftGfGfAfgagttcttL96(SEQ ID NO: 6), or asAfsgaaCfuCfUfccacUfgGfagcscsc (SEQ ID NO: 7).

Clause 16: The method of any one of clauses 5-13, wherein one or bothstrands of the RNAi agent comprises one or more dT residues at its 3′end.

Clause 17: The method of any one of clauses 5-13, wherein one or bothstrands of the RNAi agent comprises one or more modified bases.

Clause 18: The method of clause 17, wherein one or both strands of theRNAi agent is chemically modified at its 3′ end with L96(N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol(Hyp-(GalNAc-alkyl)₃).

Clause 19: The method of clause 2 or 3, wherein a RNAi reagent comprisesa sense or antisense strand as depicted in FIG. 7A.

Clause 20: The method of any one of clauses 1-19, wherein an RNAi agentis used for knocking down or inhibiting expression of an HNF4α-P2isoform mRNA, and wherein the target sequence of the RNAi agent is ahuman sequence.

Clause 21: The method of any one of clauses 1-20, wherein an RNAi agentis used for knocking down or inhibiting expression of an HNF4α-P2isoform mRNA, and wherein the RNAi agent is delivered parenterally to apatient.

Clause 22: The method of any one of clauses 1-21, wherein liver cellsare targeted for knock-down of an HNF4α-P2 isoform mRNA.

Clause 23: The method of any one of clauses 1-22, for treatment of apatient, wherein the patient is a human.

Clause 24: The method of any one of clauses 1-23, wherein an iRNAireagent is used to knock down expression of an HNF4α-P2 isoform mRNA inthe patient.

Clause 25: The method of clause 24, wherein the expression of anHNF4α-P1 isoform mRNA in the patient is not substantially affected byknocking down expression of the HNF4α-P2 isoform mRNA in the patient.

Clause 26: The method of any one of clauses 1-25, further comprisingadministering to the patient, or contacting the cells with, a secondactive agent for treatment of liver damage or liver failure in apatient.

Clause 27: A RNAi agent targeting exon 1D of HFN4α.

Clause 28: The RNAi agent of clause 27, targeting 15-30 bases of bases1-63 of SEQ ID NO: 1.

Clause 29: The RNAi agent of clause 27, comprising a sense strand havingthe sequence of at least 15 contiguous bases ofGCTCCAGTGGAGAGTTCTTACGACACG (SEQ ID NO: 1, bases 32-58).

Clause 30: The RNAi agent of clause 29, wherein the sense strand has thesequence GCTCCAGTGGAGAGTTCTT (SEQ ID NO: 1, bases 32-50),CTCCAGTGGAGAGTTCTTA (SEQ ID NO: 1, bases 33-51), TCCAGTGGAGAGTTCTTAC(SEQ ID NO: 1, bases 34-52), or GGAGAGTTCTTACGACAC (SEQ ID NO: 1, bases40-57).

Clause 31: The RNAi agent of any one of clauses 27-30, wherein one orboth strands of the RNAi agent comprises one or more, e.g., two, dTresidues at its 3′ end.

Clause 32: The RNAi agent of any one of clauses 27-30, wherein one orboth strands of the RNAi agent comprises one or more modified bases.

Clause 33: The RNAi agent of clause 32, wherein the sense strand has thesequence: GCTCCAGTGGAGAGTTCTTdTdT (SEQ ID NO: 30),CTCCAGTGGAGAGTTCTTAdTdT (SEQ ID NO: 32), TCCAGTGGAGAGTTCTTACdTdT (SEQ IDNO: 34), or GGAGAGTTCTTACGACACdTdT (SEQ ID NO: 36).

Clause 34: The RNAi agent of any one of clauses 27-30, wherein the RNAiagent is chosen from GCTCCAGTGGAGAGTTCTTdTdT (SEQ ID NO: 30),CGTGTCGTAAGAACTCTCCdTdT (SEQ ID NO: 31), gscstccaGftGfGfAfgagttcttL96(SEQ ID NO: 6), or asAfsgaaCfuCfUfccacUfgGfagcscsc (SEQ ID NO: 7).

Clause 35: The RNAi agent of clause 27, comprising a sense or antisensestrand as depicted in FIG. 7A.

Clause 36: The RNAi agent of any one of clauses 27-30, wherein the RNAiagent is chemically-modified.

Clause 37: The RNAi agent of clause 36, wherein one or both strands ofthe RNAi agent is chemically modified at its 3′ end with L96(N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol(Hyp-(GalNAc-alkyl)₃).

Having described this invention, it will be understood to those ofordinary skill in the art that the same can be performed within a wideand equivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any embodiment thereof.References incorporated herein by reference are incorporated for theirtechnical disclosure and only to the extent that they are consistentwith the present disclosure.

1. A method of treating a patient having liver damage or liver failure,comprising knocking down or inhibiting expression of a hepatocytenuclear factor 4 alpha mRNA transcribed from its P2 promoter (HNF4α-P2isoform mRNA) in the patient, or reducing activity of the proteinencoded by the HNF4α-P2 isoform mRNA, thereby treating the liver damageor liver failure in the patient.
 2. The method of claim 1, wherein theliver damage or liver failure is acute liver failure or is associatedwith alcohol-related liver disease (ALD), alcoholic hepatitis (AH), orAcute-on-Chronic Liver Failure (ACLF).
 3. The method of claim 1, whereinthe treatment normalizes or decreases blood bilirubin levels, normalizesor decreases prothrombin time, and/or normalizes or increases serumalbumin.
 4. The method of claim 1, wherein an RNAi agent for knockingdown or inhibiting expression of an HNF4α-P2 isoform mRNA isadministered to the patient in an amount effective to treat the liverdamage or liver failure in a patient.
 5. The method of claim 4, whereinthe RNAi agent targets a contiguous sequence of 15 or more bases withinthe sequence of bases 1-63 of SEQ ID NO: 1, bases 1-53 of SEQ ID NO: 1,GCTCCAGTGGAGAGTTCTTACGACACG (SEQ ID NO: 1, bases 32-58),GCTCCAGTGGAGAGTTCTT (SEQ ID NO: 1, bases 32-50), CTCCAGTGGAGAGTTCTTA(SEQ ID NO: 1, bases 33-51), TCCAGTGGAGAGTTCTTAC (SEQ ID NO: 1, bases34-52), or GGAGAGTTCTTACGACAC (SEQ ID NO: 1, bases 40-57).
 6. The methodof claim 4, wherein the RNAi agent comprises an oligonucleotide asdepicted in FIG. 7A, such as GCTCCAGTGGAGAGTTCTTdTdT (SEQ ID NO: 30),CGTGTCGTAAGAACTCTCCdTdT (SEQ ID NO: 31), CTCCAGTGGAGAGTTCTTAdTdT (SEQ IDNO: 32), TCCAGTGGAGAGTTCTTACdTdT (SEQ ID NO: 34), GGAGAGTTCTTACGACACdTdT(SEQ ID NO: 36), gscstccaGftGfGfAfgagttcttL96 (SEQ ID NO: 6), orasAfsgaaCfuCfUfccacUfgGfagcscsc (SEQ ID NO: 7).
 7. The method of claim4, wherein one or both strands of the RNAi agent comprises one or moremodified bases.
 8. The method of claim 4, wherein expression of anHNF4α-P1 isoform mRNA in the patient is not substantially affected byknocking down expression of the HNF4α-P2 isoform mRNA in the patient. 9.The method of claim 4, wherein the RNAi agent is administeredparenterally.
 10. The method of claim 1, further comprisingadministering to the patient a second active agent for treatment ofliver damage or liver failure in a patient. 11-16. (canceled)
 17. A RNAiagent targeting exon 1D of HFN4α.
 18. The RNAi agent of claim 17,comprising a contiguous sequence of 15-30 bases within the sequence of:bases 1-63 of SEQ ID NO: 1, bases 1-53 of SEQ ID NO: 1,GCTCCAGTGGAGAGTTCTTACGACACG (SEQ ID NO: 1, bases 32-58),GCTCCAGTGGAGAGTTCTT (SEQ ID NO: 1, bases 32-50), CTCCAGTGGAGAGTTCTTA(SEQ ID NO: 1, bases 33-51), TCCAGTGGAGAGTTCTTAC (SEQ ID NO: 1, bases34-52), or GGAGAGTTCTTACGACAC (SEQ ID NO: 1, bases 40-57).
 19. The RNAiagent of claim 17, comprising an oligonucleotide as depicted in FIG. 7A,such as GCTCCAGTGGAGAGTTCTTdTdT (SEQ ID NO: 30), CGTGTCGTAAGAACTCTCCdTdT(SEQ ID NO: 31), CTCCAGTGGAGAGTTCTTAdTdT (SEQ ID NO: 32),TCCAGTGGAGAGTTCTTACdTdT (SEQ ID NO: 34), GGAGAGTTCTTACGACACdTdT (SEQ IDNO: 36), gscstccaGftGfGfAfgagttcttL96 (SEQ ID NO: 6), orasAfsgaaCfuCfUfccacUfgGfagcscsc (SEQ ID NO: 7).
 20. The RNAi agent claim17, wherein one or both strands of the RNAi agent comprises one or moremodified bases.